final report bsi pas 100:2011 plant response test and herbicide sensitivity testing of...

Final report

BSI PAS 100:2011 plant response

test and herbicide sensitivity

Summary of investigations and findings into crop sensitivity to synthetic auxin herbicides, the selection of bioassay species suitable for use in the BSI PAS 100:2011 plant response test, and measures to ensure the test is sufficiently robust to deal with composts of high electrical conductivity

Project code: OAV035-003 Date: August 2014 Research date: October 2010 – July 2011

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Written by: Paul Waller

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BSI PAS 100:2011 plant response test and herbicide sensitivity 1

Executive summary

Background to Research The BSI PAS 100:2011 plant growth test using tomato is designed to demonstrate two aspects of compost quality: lack of weed seeds and other propagules, and an absence of herbicides or other chemical residues (such as volatile organic acids that are associated with inadequate composting) which would impair the growth of tomato and other plants. Following reports from the USA and New Zealand etc. on the accidental contamination of composts with residues of the herbicide clopyralid, WRAP required the BSI PAS 100 plant growth test to be reviewed to check that it was:

Sufficiently sensitive to detect traces of persistent herbicides in compost (including aminopyralid, clopyralid and triclopyr1) at concentrations that could prove damaging to sensitive plants when the composts are used in different markets (for example: use as a soil improver in agriculture and field horticulture or use in growing media); and

That this test remains fit for purpose; delivering the required sensitivity to herbicide residues whilst accommodating composts with higher than average salt contents (such as those partially derived from food wastes), ideally in a single test.

Methodology In order to address these requirements the research team first undertook two parallel series of desk exercises and range-finding experiments to:

Investigate crop and candidate bioassay species’ sensitivity to synthetic auxin herbicides;

Investigate the suitability of selected bioassay species for use in the BSI PAS 100:2011 growth test; and

Investigate the potential for raising the electrical conductivity (EC) of the peat based growing media (PBGM) used in the bioassay test through the addition of potassium chloride (KCl) as an alternative to dilution of composts with high ECs.

Based on the findings from these preliminary studies a validation trial was devised. This compared tomato with the two best candidate bioassay species (field bean and sunflower) in a BSI PAS 100:2011 plant response test with fixed 3:1 mixes of peat:compost, using composts that had a range of ECs up to almost 2100 µs/cm. These compost blends were spiked with low concentrations of clopyralid to provide a known range of herbicide concentrations of 0-333ppb. PBGM controls were included to which KCl had been added at 0, 1.0 and 1.5g/l. Conclusions and recommendations 1. The use of tomato in the BSI PAS 100:2011 plant growth test does not achieve the

objective of ensuring the detection of clopyralid in compost at sufficiently low concentrations, particularly where composts are intended for use with sensitive plants (see below). However, for the establishment of general ‘fitness for purpose’ this species should be retained for the time being.

2. Field bean (Vicia faba cv. Fuego) was effective at detecting clopyralid and some other synthetic auxin herbicide residues, even at critical low concentrations in compost, and is the most appropriate plant to use for this test. However, until more experience is gained with this species/cultivar under a wider range of conditions it should be used initially alongside tomato as a qualitative observational crop only (possibly just a single tray or pot to limit costs).

1 Triclopyr was not evaluated experimentally as the desk study established that this herbicide was rapidly degraded during

composting.


3. Species in the following key plant families are amongst those most sensitive to clopyralid and/or aminopyralid (and other synthetic auxin herbicides):

a. Asteraceae or Compositae (daisy family) b. Chenopodiaceae (beet family) c. Fabaceae or Leguminosae (pea and bean family) d. Solanaceae (potato and tomato family) e. Umbelliferae (carrot family)

4. Care must be particularly exercised to avoid using contaminated compost in the following applications:

a. In all protected and ornamental horticulture applications - as a very large range of highly sensitive plants (within and without the principal families listed above) are grown in soil and growing media situations where compost is incorporated or used as a constituent of the media.

b. In establishing clover leys or swards where clover is a valued constituent. c. In rotational cropping where a sensitive crop will follow a tolerant one.

5. It was not possible to make definitive recommendations to accommodate compost with high ECs in the BSI PAS 100 test, since the test results were confused by seasonal factors. Nevertheless, consideration should be given to fixing the ratio of peat:compost in the BSI PAS 100 test at 3:1 and, for tomato only, adding KCl to the peat-based growing medium control (at 1.0g/l) under poor growing conditions in winter, when salt stress associated with high EC composts is most disadvantageous to tomato plant germination and growth.

6. For the bioassay to be meaningful it is essential that samples of compost submitted for testing are truly representative of the batch sampled. Composters should be reminded that it is therefore essential that they follow the sampling protocol set out in AfOR’s ‘Guidelines for Compost Sampling’ which accords with BS EN 12579 ‘Soil improvers and growing media - Sampling’.

Suggestions for further work include: 1. Development of field bean as a single test species for both quantitative and qualitative

aspects of the BSI PAS 100 plant response test – to replace tomato. 2. Investigation of the response of field bean to composts of known levels of maturity and

volatile organic acid content, to establish the impacts of these variables on the plants under test, and to determine whether field bean demonstrates characteristic diagnostic symptoms for these variables in addition to the symptoms known to be associated with herbicide residues.


Contents

Glossary .................................................................................................................. 5 1.0 Introduction ................................................................................................. 6 2.0 Crop and bioassay species’ sensitivity to synthetic auxin herbicides ........... 7

2.1 Desk Studies .............................................................................................. 7 2.1.1 Review of Dow AgroSciences’ herbicide information ............................ 7 2.1.2 Review of relevant information in ‘Clopyralid Final Report’ for WRAP by

Gilbert et al (2009) ........................................................................... 9 2.1.3 Estimating potential clopyralid concentrations in UK green compost ... 10

2.2 Experimentation ....................................................................................... 10 2.2.1 Levington M2 peat-based substrate ................................................. 11 2.2.2 Levington M2 and compost mixes .................................................... 12 2.2.3 Aqueous solutions .......................................................................... 13 2.2.4 Sunflower sensitivity to clopyralid only – in Levington M2 .................. 13

3.0 Investigations into the suitability of candidate bioassay species for use in the BSI PAS 100:2011 growth test and mechanisms for dealing with composts with high ECs........................................................................................................ 14

3.1 Desk Studies ............................................................................................ 14 3.1.1 Basis for using potassium chloride (KCl) to increase the EC of the peat-

based growing medium (PBGM) control when testing composts with high ECs ........................................................................................ 14

3.1.2 Candidate bioassay species’ characteristics and their salt tolerance .... 15 3.2 Experimental work: Investigation of the suitability of herbicide sensitive species

as alternatives to tomato for use in the BSI PAS 100:2011 plant response test .............................................................................................................. 16 3.2.1 Investigation of the effects of EC, K and Cl content of a PBGM control

on the growth of tomatoes in the BSI PAS 100:2011 growth test ....... 19 3.2.2 Investigation into the effects on the growth of tomato in the BSI PAS

100:2011 growth test as a result of varying rates of dilution of composted green waste samples with a range of EC levels by peat .... 21

3.2.3 Sunflower ...................................................................................... 23 3.3 Validation trial.......................................................................................... 23

3.3.1 Species ......................................................................................... 24 3.3.2 Herbicide rates .............................................................................. 24 3.3.3 Growing media .............................................................................. 25 3.3.4 Experimental design ....................................................................... 25 3.3.5 Method ......................................................................................... 25 3.3.6 Assessments .................................................................................. 26 3.3.7 Results and discussion .................................................................... 26 3.3.8 Conclusions ................................................................................... 32

4.0 Discussion................................................................................................... 33 4.1 What are sensitive crops and applications? ................................................. 33 4.2 What is the best bioassay species to use? .................................................. 34 4.3 Composts with high EC ............................................................................. 34

5.0 Overall conclusions & recommendations .................................................... 35 5.1 Objective 1 .............................................................................................. 35 5.2 Objective 2 .............................................................................................. 36

6.0 Further work ............................................................................................... 37 7.0 Photography ............................................................................................... 37 Appendix 1 ........................................................................................................... 38 Appendix 2 ........................................................................................................... 41 Appendix 3 ........................................................................................................... 43


Appendix 4 ........................................................................................................... 46 Appendix 5 ........................................................................................................... 47 Appendix 6 ........................................................................................................... 48 Appendix 7 ........................................................................................................... 49 Appendix 8 ........................................................................................................... 51 Appendix 9 ........................................................................................................... 52 Appendix 10 ......................................................................................................... 53 Appendix 11 ......................................................................................................... 54 Appendix 12 ......................................................................................................... 55 Appendix 13 ......................................................................................................... 56 Appendix 14 ......................................................................................................... 57 Appendix 15 ......................................................................................................... 58


Glossary AfOR Association for Organics Recycling2 Ca Calcium CRD Chemicals Regulation Directorate CGW Composted green waste or green compost Cl Chloride (Chlorine) cv Cultivar CV Coefficient of Variation (Standard Deviation of a mean expressed as a % of

that mean) DA Dow AgroSciences DAS Days after sowing EC Electrical Conductivity (expressed in μS/cm) HTA Horticultural Trades Association K Potassium KCl Potassium chloride LSD Least Significant Difference MC Moisture content Mg Magnesium Na Sodium NOEL No Observable Effect Level PAS Publicly Available Specification PBGM Peat-Based Growing Medium ppb Parts per billion ppm Parts per million ppt Parts per trillion S Sulphur SAH Synthetic auxin herbicide SD Standard Deviation (of a mean) STC Stockbridge Technology Centre Ltd. WRAP Waste & Resources Action Programme

2 Please note that, since the time of writing, AfOR have become the Organics Recycling Group (ORG) – part of the Renewable Energy Association (REA)


1.0 Introduction The BSI PAS 100:2011 plant growth test using tomato is designed to demonstrate two aspects of compost quality: lack of weed seeds and other propagules, and an absence of herbicides or other chemical residues (such as volatile organic acids that are associated with inadequate composting) which would impair the growth of tomato – and hence, other plants. Following reports from the USA and New Zealand etc. on the accidental contamination of composts with residues of synthetic auxin herbicide, WRAP required the BSI PAS 100 plant response test to be reviewed to check that it was:

Sufficiently sensitive to identify batches of compost that may contain crop-inhibiting levels of persistent herbicide residues (which have not been broken down during the composting process); and

Appropriate and robust enough to accommodate composts with the higher electrical conductivities which can occur through the use of food waste as compost feedstock.

In order to address these requirements the research team first undertook two parallel series of desk exercises and range-finding experiments to:

Investigate candidate bioassay species’ sensitivity to synthetic auxin herbicides (see Section 2.0); and

Investigate candidate bioassay species’ sensitivity to increased electrical conductivity (EC) (see Section 3.0).

On completion of these preliminary studies a validation trial was devised (see Section 3.3). This compared tomato, field bean and sunflower in a plant response test with 3:1 mixes of peat:compost. The EC of these mixes ranged from 673 to 2000 µs/cm, and they were spiked with clopyralid to give a range of herbicide concentrations in the compost from 0 to 333ppb. This report summarises the key points and the outcomes from the desk exercises and trials programme conducted by the research team and concludes with recommendations. Photographs of diagnostic synthetic auxin or hormone herbicide damage symptoms are presented in Appendices 8 and 9.


2.0 Crop and bioassay species’ sensitivity to synthetic auxin herbicides Prior to and in parallel with experimental work three desk exercises were carried out:

A review of confidential and non-confidential herbicide information provided by Dow AgroSciences;

A review of relevant information contained within the ‘Clopyralid Final Report’ for WRAP by Gilbert et al (2009); and

Development of a theoretical estimate of the range of clopyralid concentrations that might be found in composts in the UK.

2.1 Desk Studies 2.1.1 Review of Dow AgroSciences’ herbicide information The principal aim of this study was to ensure that appropriate herbicides, rates and test species were selected for the planned plant response trials. Dow AgroSciences (DA) manufactures the three herbicides of primary interest (aminopyralid, clopyralid and triclopyr) as well as picloram which, along with a number of other synthetic auxin herbicides, has previously been found to be responsible for economic plant damage on an ornamental nursery in the UK in 2008 – although in that case the suspected source of contamination came from outside the UK (McPherson, pers comm). DA kindly provided detailed information on their products on a confidential basis, together with useful background information on the family of synthetic auxin herbicides3. This included detailed information concerning the activity of pyridine-carboxylic acid (“synthetic auxin”) herbicides and the sensitivity of major UK crops and plant families to them, including specific ‘no-effect’ levels in soils. Other synthetic auxin herbicides which might produce similar effects and which are commonly used in amateur lawn care products include 2-4D, dicamba and MCPA. These are generic products produced by a number of suppliers and none provided any information on the impacts of these compounds on composting or compost quality during this research. Information provided by Dow AgroSciences and gleaned from other sources enabled the research team to select appropriate representatives of the three key sub-groups of the synthetic auxin herbicide (SAH) compounds for further study, namely: pyridine-carboxylic acids, phenoxy-carboxylic acids and benzoic acids. The aim was to select those herbicides which were likely to be the most active and which might show different symptom expression in the selected hosts. Whilst the plan was to select two from each group, in the case of the benzoic acids only one active was readily available, so a third herbicide from the biggest group was added – picloram. The conclusions were:

Residues of the synthetic auxin herbicides which appear to be resistant to degradation via composting or passage through animal digestive tracts are highly unlikely to have any adverse effects on crops for which they already have on- or off-label approval.

Clopyralid, aminopyralid, tricylopyr and picloram (as well as 2-4D, dicamba and MCPA) have little or no effect on monocotyledonous plants (e.g. grass or graminaceous species) so compost contaminated with residues of these compounds can probably be used in such applications without concern.

Similarly, based on approvals and experience, it is highly unlikely that residues of any of clopyralid, aminopyralid, tricylopyr and picloram will have any adverse effects on brassicas (Family Brassicaceae)4. For this reason Chinese cabbage, which is the standard bioassay species used in Germany (Gerald Schmilewski, pers comm.) and specified in a draft

3 A useful guide to the chemical relationships between the synthetic auxin and all the other herbicide groups is given in the ‘World of Herbicides’ modes of action poster; at http://www.hracglobal.com/Portals/5/moaposter.pdf 4However, it is known that brassicas can be damaged by MCPA and possibly some of the other SAH’s, but the symptoms are different from the more typical ones affecting the apex and leaves as reported here.

http://www.hracglobal.com/Portals/5/moaposter.pdf


European plant response test standard (Paul Waller, pers comm.), was not considered a prime candidate for use in a herbicide bioassay, but it was included in screening work for comparative purposes.

Crops which are exposed to contaminated compost only via mulching are less likely to be affected by herbicide residues. This is because they are established and (often) deep rooted, so they are both in a less sensitive state and less likely to encounter contamination at sufficiently high concentrations. Such crops include orchard and cane fruit and field grown strawberry – unless newly planted.

Three families of plants are known to be highly sensitive to clopyralid, aminopyralid, tricylopyr and picloram: Asteraceae (Compositae), Fabaceae (Leguminoseae) and Solanaceae; representatives of all three have been evaluated in this project as candidate bioassay species. These representatives are: dahlia, sunflower, clover, field bean, lentil, pea and tomato.

Ornamental bedding and pot plants, especially those grown under protection, are at higher risk than field-grown (non-ornamental) crops for a number of reasons:

A wide variety of highly sensitive species is grown, with the highly susceptible Asteraceae family widely represented since many of these are amongst the most popular annual flowering plants;

Compost may be incorporated directly into the growing medium at much higher rates than would be encountered in field horticulture (or agriculture);

The crops are often juvenile and in a more sensitive state;

The root zone is restricted and roots cannot escape any herbicide contamination;

They are irrigated on flood and capillary benches and as the herbicides are water soluble rapid dispersal and plant damage is possible; and

There is a risk of vapour action with some of the chemicals in a protected (enclosed and relatively high temperature) crop environment.

Relevant extracts from Dow AgroSciences’ UK product labels for pyridine-carboxylic acid herbicides summarising on-label uses, crop sensitivity data and comments on persistence and degradation provide useful information and are summarised in Appendix 1. In addition it was concluded that:

A single bioassay species was unlikely to exhibit symptoms that are unique to individual herbicides as those under study have similar modes of action. It was more likely that similar symptoms would be expressed to varying degrees, although this could also result from differences in herbicide concentration. Therefore a single-species bioassay is unlikely to be able to speciate5 any herbicide residues with confidence, and predictions of safe use will be uncertain. There may even be residues of herbicides present which produce no symptoms in the species under test within the time of the bioassay.

Crops, even in the same plant family, are not necessarily of equal sensitivity to the herbicides in question.

Therefore, it is impossible to be definitive about which individual crops may be safely exposed to compost that bioassay shows to be contaminated with one or more unknown herbicides, or to publish a definitive and defensible plant/herbicide sensitivity matrix from the information available. Such guidance can only be provided in the most general terms, and an indicative table of sensitivity to aminopyralid and clopyralid in the major crop plant families is given in Appendix 2. What is very important is a familiarity with the characteristic symptoms caused by synthetic auxin herbicides so that these can be readily recognised and appropriate action taken.

5 A sensitive host bioassay if the most effective way of determining the presence of synthetic auxin herbicides in substrates and composts at very low, but critical levels;

however, this technique does not differentiate between the different chemicals that may be responsible. Provided the concentration present is above the level of detection specified by the laboratory and the responsible herbicide(s) are included in the analysis suite, then laboratory analysis can be used to identify specific compounds. Sensitivity can be improved by requesting a leachate analysis on the substrate as this generally improves the sensitivity of the test.


Picloram and triclopyr are approved for use on land ‘not intended for cropping or vegetation’ and grass treated with the former should not be composted. Triclopyr is also approved on agricultural grassland and as a home and garden product. However, work on triclopyr published in 2007 by the Recycled Organics Unit in New South Wales Australia has demonstrated that it does not persist through the composting process6 and, accordingly, it was excluded from further work. 2.1.2 Review of relevant information in ‘Clopyralid Final Report’ for WRAP by Gilbert et al

(2009) Whilst this report does not claim to be exhaustive, it does represent an authoritative investigation of key issues relating to problems that have been associated with the composting of, principally, grass material treated with herbicides containing clopyralid and aminopyralid (and any associated herbicides also present in the formulated ‘mixed’ products sold in the UK such as fluroxypyr and MCPA). In the UK, whilst it is possible for both of these herbicides to enter the commercial composting streams (through feedstocks such as livestock manures and grass clippings), it is clopyralid that is of greater interest as it is the main or a common constituent of amateur lawn herbicides and – although products now all include clear warnings to consumers on the label not to compost treated grass clippings from the first four cuts (nor to put these clippings in organics recycling bins) – there remains a potential risk of contamination from a large number of uncontrolled domestic sources. Aminopyralid is only available for professional use under a stewardship scheme, and treated material should not leave the field where the compound was applied. Gilbert et al concluded that: ‘The predominant factor with clopyralid (and similar chemicals) in compost is not its rate of decomposition. In general, clopyralid seems to decompose fairly well in the typical time frame of a composting process (although the Australian studies apparently suggest otherwise).’…..‘Clopyralid in compost is a story about concentration and coincidence; concentration because of clopyralid potency at very low concentrations (and that of like products); coincidence because tainted compost must coincide with particular uses to become a problem.’ In summary they stated ‘Clopyralid degradation during composting is highly variable’ and ‘short composting periods may not be sufficient to degrade clopyralid should it be present in composting feedstocks. However, as most compost applied to fields would be ploughed prior to planting crops, it seems likely that potential contamination would present a minimal risk. Where compost has been used as a mulch (for example, in the production of top fruit) no incidences of herbicide effects have been documented.’ In the report there are a number of figures quoted for the concentrations of clopyralid found in various US samples when problems were at their peak about ten years ago, and the levels at which plants have demonstrated sensitivity (all presumably on a dry mass basis):

In Table 8, concentrations ranging from a trace to >200ppb were reported in several US states between 1999 and 2002;

On one occasion 1550ppb was found in grass clippings and 477ppb in immature compost;

Concentrations of 70-150ppb were found in spring feedstocks dominated by grass clippings in Washington State; and

Vulnerable plants e.g. tomato are said to be sensitive to clopyralid concentrations at/or below 1-10ppb.

6 See http://www.recycledorganics.com/publications/reports/riskmgtprog/persistherbRMP.htm

http://www.recycledorganics.com/publications/reports/riskmgtprog/persistherbRMP.htm


On the subject of bioassays, the report cites a number of test species that might be used for BSI PAS 100 as alternatives to the current tomato (Lycopersicon esculentum cv. Shirley (F1)):

In the UK, Dow AgroSciences suggest gardeners use broad bean (Vicia faba – no variety specified – equivalent to field bean)7;

Washington State University suggested gardeners and researchers use garden peas (Pisum sativum, no variety specified)8; and

Red clover (Trifolium pratense) is recommended by US9 and Australian10 experts as an alternative to tomato (Lycopersicon esculentum), since it is highly sensitive to clopyralid and produces observable results in 14 days.

2.1.3 Estimating potential clopyralid concentrations in UK green compost Clopyralid is the synthetic auxin herbicide of greatest interest because it is used in many domestic turf products, albeit alongside other herbicides, and is known to persist through the composting process. Therefore a theoretical exercise was conducted by the research team with input from the Scotts Company UK Ltd. (one of the manufacturers of retail lawn products based on clopyralid) and AfOR to model potential levels of clopyralid contamination of composted green waste (CGW) via treated domestic lawn clippings, based on a variety of expert assumptions. The aim was to estimate the possible range of concentrations that might be found in the finished compost products – although the lowest possible concentration would always be zero. Details are given in Appendix 3. The results suggested that if there was to be extreme contamination (highest recommended rate of clopyralid application, 100% capture of the applied herbicide by the treated grass, one grass cut of just 30g/m2 to remove all herbicide, 50% grass in any one composting batch and no herbicide degradation during composting) then the concentration of clopyralid in CGW on a dry weight basis might, in this ‘absolute worst case’ scenario, be around 1000ppm – but this is not realistic and such a figure is in the order of 1000 times the actual maximum detected in US samples. The ‘realistic worst case’ scenario suggests that the concentration encountered would probably not exceed 861ppb and could even be below 1ppb. This range compares better with the actual levels from the US data reported by Gilbert et al (2009) above. 2.2 Experimentation The current BSI PAS 100 test uses tomato plants to ensure composts are ‘fit for purpose’, including assurance of freedom from residual persistent herbicides. The primary purpose of this element of the project was to evaluate the relative sensitivity of different host species (in comparison with tomato) to a range of synthetic auxin or’ hormone’ herbicides applied at different concentrations through the use of compost samples spiked with known concentrations of herbicide. Four different experiments on a range of plant species were conducted in the following substrates:

A proprietary peat-based substrate (Levington M2) in pots – simulating the peat-based growing medium (PBGM) used in the BSI PAS 100:2011 test;

A range of Levington M2+CGW mixes in pots – simulating the test mixes used in the BSI PAS 100:2011 test;

Aqueous solutions of known herbicide concentration in vials; and

A small-scale test in Levington M2 using only sunflower as the host.

7 See http://www.dowagro.com/uk/mark-up/faqs.htm 8 Washington State University and Washington State Department of Ecology (2002) Bioassay Test for Herbicide Residues in Compost: Protocol for Gardeners and Researchers in Washington State. Information Sheet published online at: http://www.puyallup.wsu.edu/soilmgmt/Pubs/CloBioassay.pdf 9 Brinton, W.F., Evans, E. and Blewett T.C. (2005). Herbicide Residues in Composts: pH and Salinity Affect the Growth of Bioassay Plants. Bull. Environ. Contam. Toxicol.

75:929-936 10 Recycled Organics Unit (2007a). Persistent Herbicides Risk Management Program: Research Report and Recommended Action Plan. Second Edition. Recycled Organics Unit, internet publication at: http://www.recycledorganics.com/publications/reports/riskmgtprog/persistherbRMP.htm

http://www.dowagro.com/uk/mark-up/faqs.htm

http://www.puyallup.wsu.edu/soilmgmt/Pubs/CloBioassay.pdf

http://www.recycledorganics.com/publications/reports/riskmgtprog/persistherbRMP.htm


Of the candidate bioassay species, clover was not tested because of its growth habit and difficulty of growing single plants. Sunflower was added later and was tested on its own. 2.2.1 Levington M2 peat-based substrate Tomato, dahlia, Chinese cabbage, pea & lentil were raised from seed in module trays and transplanted singly into 9cm pots of substrate in preparation for this study. Field bean seeds were sown directly into the 9cm pots, with the sowing of all six species being scheduled to try to ensure the different species attained the 1st true leaf stage simultaneously. Due to changeable environmental/ climatic conditions the reality was slightly different and the trial had to be run in parallel with the various species being treated at different times. Three-replicates were sown for each species. The synthetic auxin herbicides were applied to each pot as a 10ml aliquot poured onto the compost surface of each pot at the 1st true leaf stage. The synthetic auxin herbicides aminopyralid, clopyralid, picloram, dicamba, MCPA & 2,4-D were each prepared as 10-fold dilution series and applied at 1000ppm, 100ppm, 10ppm, 1ppm, 100ppb, 10ppb, 1ppb, 100ppt, 10ppt, 1ppt, and 0ppm as a water control. Following treatment application, the pots were irrigated overhead to ensure an even infiltration of the water soluble products into the growing medium. All pots (ca. 1100) used for this study were placed in saucers (to avoid any loss of herbicide) on twin benches lined with polythene to capture any run-off and hence avoid any contamination risk in the glasshouse. The plants were then irrigated periodically to maintain field capacity but from below, via the saucers, to retain all the applied herbicide in each container (pot) unit. The plants were monitored for characteristic and other unusual symptoms over a 4-6 week period; detailed assessments were made at 7-day intervals for 28 days and as required thereafter. In this pot study the first symptoms of herbicide damage became apparent within 24 to 48 hours at the highest treatment dose rates, though at lower rates the symptoms were slower to appear. Detailed interim assessments were made at 7, 14 & 21 days (data not published), with the results after 28 days described in this report and summarised in Appendix 3. Host susceptibility: There was a considerable difference in the degree of symptom expression between the different species selected for study. The brassica (Chinese cabbage) was very tolerant and insensitive to even high doses of the various herbicides evaluated and at best showed slight distortion of the youngest leaves at 1000 to 100ppm. At rates below 100ppm symptoms were largely absent. In the case of clopyralid it was difficult to find any adverse symptoms in this species – even at the highest doses applied. Any symptoms that did occur in Chinese cabbage were not particularly diagnostic11. Dahlia, tomato & field bean exhibited the most characteristic symptoms, with severe distortion of the growing point(s) and also in-rolling of the younger leaves. At lower rates tomato leaves became spiky (like nettles). At high dose rates the plants were either severely stunted or killed. Pea seedlings were also severely affected at the highest dose rates and were either stunted or killed. At lower dose rates characteristic distortion of the growing points was not particularly apparent and the main symptom was an inward rolling of the young leaves. The symptoms expressed in pea plants were not regarded as particularly diagnostic compared with those in dahlia, tomato or field bean.

11 It is interesting to note that Chinese cabbage & other cruciferous hosts are routinely used in some countries e.g. Germany as a bioassay species of choice. Based on

the results of this experiment, the use of Chinese cabbage for the detection of synthetic auxin herbicides is clearly inappropriate.


The lentil plants initially appeared to be the most sensitive of all the species tested as in-rolling of the leaves was very evident, even at extremely low concentrations. At high dose rates lentil seedlings were stunted or killed and whilst some characteristic shoot distortion symptoms did develop they were not particularly consistent and the delicate nature of the lentil shoots made it difficult to observe the symptom clearly. Unfortunately, the in-rolling of the lentil leaves was also visible on some of the untreated control plants in the study and again this symptom was not considered sufficiently diagnostic for routine use. Herbicide dose – No Observable Effect Level (NOEL): Overall, it was evident that the level of sensitivity of the various plant species to the different synthetic auxin herbicides was less than expected and was, in general, around 10 to 100ppb (with the exception of Chinese cabbage). Previous confidential studies at STC using dahlia had shown seedling sensitivity in dahlia as low as 250ppt (the lowest concentration tested) (McPherson, pers comm.). It is therefore important in this respect to consider that the earlier dahlia studies were conducted during the main summer growing season when temperatures and solar radiation levels were high, whereas the current study was undertaken during the cooler period of February to March when solar radiation levels were much reduced. It is not known whether this may have an impact on the plant availability of the various synthetic auxin herbicides or seedling sensitivity but, as results here suggest a No Observable Effect Level (NOEL) of around 10 to 100ppb, caution is needed as a different sensitivity profile may occur at other times of the year. Conclusions:

No single host species stood out as being ‘ideal’ for inclusion in the final validation study, though compared with the standard host tomato, both dahlia & field bean exhibited the most diagnostic characteristics at the lowest levels of sensitivity to the most herbicides.

Unfortunately, module-raised dahlia seedlings were considered impractical for routine use in a BSI PAS 100:2011 bioassay. It was however considered that another member of the Asteraceae family, particularly sunflower (which has large seeds and is therefore easy to handle), may offer an alternative solution and should be evaluated12.

Lentil, which initially looked promising in terms of sensitivity, showed relatively poor diagnostic characteristics and extensive in-rolling of the leaves. This, coupled with its irregular branching habit has led us to reconsider the suitability of this species for the BSI PAS 100:2011 growth test.

Chinese cabbage was clearly insufficiently sensitive to be of value in a herbicide bioassay.

Pea, whilst more sensitive than Chinese cabbage, did not show characteristic symptoms like tomato, dahlia or field bean and is of no further value in the validation stage. The tendrils also became very tangled and made their use in trays problematic.

Whilst the NOEL was higher than expected in tomato plants and given that dahlia was not suitable (as plug plants), field bean was considered most appropriate as an alternative test species.

2.2.2 Levington M2 and compost mixes The herbicides of concern all bind to organic matter such as celluloses (in the soil or test substrate) and the rate or ease with which it is released into solution has a profound effect on its availability to plants via their roots. The first experiment (Section 2.2.1) was conducted in an entirely peat-based substrate (simulating the PBGM used in the BSI PAS 100:2011 growth test) but compost may have a different binding capacity than peat. In the PAS bioassay, compost samples are tested in admixture with peat, typically at 1:3 (or 25% compost + 75% peat) which reduces the EC of the test material to a level that permits tomato seed germination and growth, but it should

12Subsequently, the research team undertook a series of small-scale studies with direct-sown sunflower to assess if it might be usefully be included in the validation stage

of the study.


be noted that such a dilution also reduces the concentration of herbicide in the test substrate – in this case by a factor of four. This cannot be avoided. But, whilst at high levels of contamination this may not be critical, at a low level the concentration might be reduced below the NOEL for the species being used in the bioassay. To evaluate the effect of organic matter type (or the ratio of CGW:peat in the substrate) on symptom expression, a separate study was conducted using clopyralid – as the most commonly used active substance in amenity/garden scenarios – at a range of dilutions (10ppm, 1ppm, 10ppb, 1ppb & 0ppb control) with the same six host species in a range of CGW to peat substrate (Levington M2) mixtures (50:50; 37.5:62.5; 25:75; 17.5:82.5; & 0:100). This study was conducted using six replicates alongside the host sensitivity study but on a different bench in the same glasshouse. The plants were monitored for characteristic and other unusual symptoms at 7-day intervals for 28 days. The 28-day data are summarised in Appendix 4. Results and Conclusions:

The results for the 0% CGW treatment (i.e. 100% Levington M2) are, as expected, virtually the same as those obtained in the previous experiment with clopyralid;

There was no consistent effect on crop sensitivity to clopyralid when peat was replaced by up to 50% CGW in the substrate across the range of host species. This suggests that there is no major difference in the binding capacity of the two substrates for clopyralid (on a volumetric basis);

Furthermore, using peat as a diluent in the BSI PAS 100 growth test will have no detrimental effect on the availability of clopyralid residues in CGW for plant uptake or on the symptoms expressed – beyond the simple physical effect of reducing the overall concentration in the test substrate;

The NOEL was again unexpectedly high compared to other confidential studies conducted with dahlia; and

The physical dilution of any pesticide residues in the CGW substrate caused by the (necessary) addition of peat may adversely impact on the overall sensitivity of the test.

2.2.3 Aqueous solutions This short-term study was a duplicate of the first experiment (Section 2.2.1), using host seedlings in an aqueous solution in sterile vials, providing rapid results from a single assessment made after seven days. The full table of results is included at Appendix 5. Results and conclusions:

The results were largely comparable with those obtained in the first experiment, but the plants showed slightly more sensitivity to the herbicides;

This procedure had the advantage that it is quicker to establish and takes up significantly less space than pot tests. The disadvantage was that the seedlings suffered adversely under the artificial lighting regime used in the glasshouse during late winter conditions, and only remained viable for 7 to 10 days, so the test had to be terminated early;

Further development of this approach would be required to substitute for the BSI PAS 100 tray test; but

The vial study does indicate the potential for a new approach, to test leachate samples from compost stacks. In theory this might minimise the problem of substrate sampling and the potential to miss herbicide ‘hot-spots’ in compost – although it does rely on leachate generation, which would normally be minimized in composting processes to reduce the need for specific leachate management interventions.

2.2.4 Sunflower sensitivity to clopyralid only – in Levington M2 A late, small-scale study with sunflower (an alternative to dahlia in the family Asteraceae) was established with seed direct-sown into substrate drenched with herbicide solutions of


various strengths. Contrary to literature indications, this species did not exhibit particularly characteristic symptoms or diagnostic responses to critical low levels of synthetic auxin herbicide within the test period. Nevertheless, at the highest dose rate (1ppm), emergence was poor and the hypocotyls of those seedlings that emerged were slightly twisted and distorted; but these symptoms per se are not sufficiently diagnostic of herbicides in this group. 3.0 Investigations into the suitability of candidate bioassay species for use in

the BSI PAS 100:2011 growth test and mechanisms for dealing with composts with high ECs

Prior to any experimental work, two desk exercises were carried out:

An assessment of the practicability of using any of the candidate bioassay species and their salt tolerance – given that it was required that the chosen species must provide robust data when exposed to composts with high EC; and

An assessment of the possibility of using potassium chloride (KCl) to increase the EC of the peat-based growing medium (PBGM) controls instead of diluting high EC composts to a greater extent than the 4:1 maximum specified in the current procedure.

3.1 Desk Studies 3.1.1 Basis for using potassium chloride (KCl) to increase the EC of the peat-based growing

medium (PBGM) control when testing composts with high ECs In its submission to WRAP, the research team proposed that instead of (or perhaps as well as) changing the peat dilution ratio when testing CGWs with high EC, it might be appropriate to increase the EC of the PBGM using KCl to make it a more valid control. The basis for this was an analysis of data provided by AfOR of the results of compost analyses generated through its Compost Certification Scheme in 2009. These data (excluding a few obvious outliers – samples where nutrient levels and EC were not reconcilable) are presented in Figure 3-1, whilst the mathematical relationships between these six elements and EC are shown in Table 3-1.

Figure 3-1 Relationship between compost water-soluble nutrient levels and EC (in μS/cm)

0

500

1000

1500

2000

2500

3000

3500

4000

0 500 1000 1500 2000 2500 3000 3500

mg/l (

wa

ter

solu

ble

)

EC

K

Ca

Mg

S

Cl

Na

Linear(K)


Table 3-1 Correlation between the water-soluble nutrient content of six elements in composts and their EC values

Water Soluble Element

Correlation Coefficient

Slope Intercept

K 0.878 1.134 118

Cl 0.724 0.593 187

S 0.663 0.151 -54

Ca 0.657 0.088 -1

Na 0.582 0.134 34

Mg 0.181 0.018 10

This statistical analysis confirms that increases in the EC of composts are primarily and most closely correlated with increases in their water-soluble K and Cl contents; and (surprisingly) not related to increases in Na content to the same degree. Thus, adding KCl to the PBGM should most closely mimic the cause(s) of increased EC in compost. It was calculated that rates of up to 3g/l would be appropriate. 3.1.2 Candidate bioassay species’ characteristics and their salt tolerance The current BSI PAS 100:2011 growth test requires the quantitative determination of germination and plant mass from a known number of sown (tomato) seeds together with sensitivity to low concentrations of ‘herbicides of concern’ within 28 days or less. It is AfOR’s preference (Emily Nichols, pers comm.) that any revised test procedure should involve a single test species that is sensitive to low concentrations of ‘herbicides of concern’ and that can provide robust quantitative as well as qualitative assessments of plant response irrespective of the EC of the compost under test (up to 2000μS/cm or more). Accordingly, the research team determined that any alternative to tomato for use in the current format of a growing test in half-trays should have the following characteristics:

Have seeds large enough to be handled and counted easily;

Be fast growing from directly sown seed under the current conditions of the BSI PAS 100:2011 test;

Have a habit that is upright and uniform; and

Be tolerant of appropriately high conductivity levels without displaying symptoms that may be confused with herbicide contamination.

And also:

Be highly sensitive to low levels of the synthetic auxin herbicides such as clopyralid; and

Quickly display easily recognised and characteristic symptoms of hormone herbicide damage.

Based on research and agronomic knowledge within the research team, the relevant agronomic characteristics of each of the potential bioassay plants were assessed and the results summarised in Table 3-2.


Table 3-2 Comparison of the agronomic characteristics of candidate bioassay species

Plant Seed size Direct sown?

Speed of growth

Growth habit Salt tolerance

Dahlia Small No, plugs only

Slow Upright Very lowb

Field bean Large Yes Fast Upright & vigorous

Lowa

Lentil Large Yes Moderately fast

Upright but irregular no. of stems & low mass

Lowb

Pea Large Yes Fast

Upright but not self –supporting Plants tangle due to tendrils

Lowa

Red clover Small Yes Moderate Lax Lowa

Tomato Medium Yes Moderately fast

Upright Moderatea

Sunflower Large Yes Fast Upright >Moderatea

a Given in Agri-Facts, November 2001 (Agdex 518-17)

http://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/agdex3303/$file/518-17.pdf?OpenElement b Research team classification based on plant family and agronomic knowledge

Based on the above evaluation it was concluded that dahlia and red clover were unsuitable for use in the BSI PAS 100:2011 growth test due to their small seed size and, in the cases of clover, its growth habit too. Three leguminous plants – field bean, lentil (a plant used in confidential trials at STC for herbicide residue evaluation) and pea should be evaluated even though their salt tolerance would appear to be lower than tomato. Belatedly, it was decided that, given the herbicide sensitivity and importance of Asteraceae in the most sensitive horticultural applications (namely bedding and pot plant production), sunflower should be assessed in small scale tests with a view to its inclusion in the validation phase as an alternative to module-raised dahlia. 3.2 Experimental work: Investigation of the suitability of herbicide sensitive species as

alternatives to tomato for use in the BSI PAS 100:2011 plant response test The three leguminous plant species (field bean, lentil and pea) identified as most promising in the desk study above were compared with tomato for their suitability for use in the BSI PAS 100 plant response test. Red clover and dahlia were excluded. Both field bean and lentil have previously been used in confidential soil herbicide residue studies at STC whilst pea has been recommended for use in a clopyralid bioassay by Washington State University.

http://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/agdex3303/$file/518-17.pdf?OpenElement


To determine if the species were suitable for use in growing media with a range of EC levels and salt concentrations, the four species were tested in three growing media based on different components:

Standard peat (PBGM) – estimated EC 284μS/cm;

Standard peat plus potassium chloride at 1.5g/litre of peat – estimated EC 736μS/cm; and

Standard peat plus CGW with an EC of approximately 1200μS/cm mixed at a ratio of 3:1 – estimated EC 490 μS/cm

Results: The germination and harvest data are summarised in Table 3-3 and Table 3-4.

Table 3-3 Germination percentage at 14 days after sowing

Species and media

Mean

SDa (+/-) CVb (%)

Tomato Peat (PBGM) Peat + KCl Peat + CGW All media Field bean Peat (PBGM) Peat + KCl Peat + CGW All media Lentil Peat Peat + KCl Peat + CGW All media Pea Peat (PBGM) Peat + KCl Peat + CGW All media

98.3 95.0 100.0 97.8 95.0 98.3 100.0 97.8 96.7 98.3 93.3 96.1 85.0 81.7 88.3 85.0

4.1 5.5 0.0 4.3 5.5 4.1 0.0 4.3 5.2 4.1 5.2 5.0 15.2 9.8 13.3 12.5

4.2 5.8 0.0 4.4 5.8 4.2 0.0 4.4 5.3 4.2 5.5 5.2 17.8 12.0 15.0 14.7

a Standard Deviation b Coefficient of variation (SD of a mean expressed as a % of that mean)

Lentil was the first species to emerge followed by pea and field bean, with tomato the slowest. The emergence of pea and field bean was slightly delayed for the peat + KCl treatment, but by nine days for pea and eleven days for field bean the emergence was similar for all three media. For tomato, emergence was significantly slower for the peat + KCl and the peat + CGW, but by eleven days they were both similar to the peat only (PBGM) control.


Table 3-4 Mean weight per plant (g) at harvest

Species and media

Mean SDa (+/-) CVb (%)

Tomato Peat (PBGM) Peat + KCl Peat + CGW All media Field bean Peat (PBGM) Peat + KCl Peat + CGW All media Lentil Peat Peat + KCl Peat + CGW All media Pea Peat (PBGM) Peat + KCl Peat + CGW All media

2.4 2.2 2.4 2.3 5.9 5.7 5.4 5.7 0.68 0.88 0.62 0.73 2.2 2.3 2.0 2.2

0.2 0.7 0.6 0.5 0.8 0.7 0.4 0.7 0.08 0.10 0.12 0.15 0.6 0.4 0.3 0.4

9.1 30.9 23.7 21.6 13.0 13.1 7.7 11.6 11.0 11.1 19.0 20.4 25.4 15.6 14.6 19.5

a Standard Deviation b Coefficient of variation (SD of a mean expressed as a % of that mean)

Plant quality for all four species was good with vigorous growth and foliage colour. Tomato and field bean growing in the peat + KCl were slightly (but not significantly) smaller than in the other media treatments. At four weeks after sowing the field beans growing in the peat plus compost had cupped leaves at the head of the plant (a symptom associated with some herbicides or stress), but the cause was not clear as this treatment did not have the highest EC. No other species or treatment in this trial exhibited these symptoms. At harvest the plant weights were highest for the field bean and lowest for the lentil, with both pea and tomato similar, although tomato had been harvested at five weeks after sowing rather than four – because growth was slowed due to the exceptional cold, snowy and cloudy conditions during much of the test (despite creating the environmental conditions recommended in the current test procedure). Importantly, plant weights in each species were similar for all three media treatments. Assessments of the variability of the results for germination and emergence and plant weights at harvest indicate that pea is the least suitable alternative species tested. Conclusions:

Field bean and lentil were the most agronomically suitable alternative species to tomato in the BSI PAS 100 growth test; and

These species would probably not be affected by higher EC levels in the test media to any greater degree than tomato.


3.2.1 Investigation of the effects of EC, K and Cl content of a PBGM control on the growth

of tomatoes in the BSI PAS 100:2011 growth test Based on the findings of the desk study reported above, batches of PBGM were prepared with the addition of potassium chloride at 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0g/l, Tomato seeds were sown on 10 November 2010 and the effects on emergence and growth were recorded over 5 weeks with the trial harvest delayed until the end of the fifth week (on 15 December 2010) due to the poor growing conditions. Results: The PBGM analysis showed that the addition of the standard rate of NPK fertiliser alone had a significant effect on raising the EC of the peat and this was further increased by increasing the rate of potassium chloride. The data are given below in Table 3-5.

Table 3-5 The effect of KCl addition rate to PBGM on substrate EC

KCl addition to PBGM

Substrate EC (μS/cm)

Mean Range

Nil (Control) 0.5g/l 1.0g/l 1.5g/l 2.0g/l 2.5g/l 3.0g/l Peat only Peat + lime

284 420 630 736 1014* 1008* 1387 45 56

274 – 303 407 – 428 621 – 646 718 – 747 987 – 1044 926 – 1052 1314 – 1444 39 – 50 54 – 59

*These means should have been significantly different from each other but despite repeated testing it was not possible to

establish sensible figures. However, the growth data provided a more coherent separation of these treatments.

From Figure 3-2 it can be seen that the emergence of the tomato seeds was delayed where potassium chloride was incorporated into the peat at above 0.5g/litre of peat. Seedling emergence for the control and 0.5g/l KCl treatments were similar, but emergence was slower at higher rates. Final plant number was similar for KCl treatments <2.5g/l, but the delay in emergence and poorer subsequent growth affected plant weights at 5 weeks (see Table 3-6).


Figure 3-2 The effect of KCl addition rate to PBGM on the tomato seedling emergence 7-23 days after sowing

Table 3-6 The effect of KCl addition rate to PBGM on the tomato plant weight at harvest (5 weeks after sowing) Five weeks after sowing, the plant weights for the control and 0.5g/l KCl were very significantly higher than the 1g/l treatment, which in turn were very significantly higher than the 1.5 and 2g/l treatments. The 2.5 and 3g/l treatments were similar and significantly smaller than the 1.5 and 2g/l rates.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

7 8 9 10 11 12 13 14 15 16 23

0 g/l

0.5 g/l

1 g/l

1.5 g/l

2 g/l

2.5 g/l

3 g/l

KCl rate

KCl addition to PBGM Mean plant weight (g)

Nil (Control) 0.5g/l 1.0g/l 1.5g/l 2.0g/l 2.5g/l 3.0g/l LSD (Least Significant Difference) (at the 99.5% confidence level)

3.06 2.80 2.17 1.61 1.31 0.85 0.54 0.335

Percentage

emergence

Days after sowing


Conclusions:

The addition of potassium chloride to the PBGM provides a practical means of mimicking the EC, K and Cl levels experienced when diluting high EC compost samples at a volume ratio of 3:1 or 4:1 with peat in the BSI PAS 100:2011 growth test; and

For tomatoes, a rate of 1.0g/l KCl should be added to the PBGM under poor growing conditions (Nov-Mar inclusive) if excessive retardation of growth is to be avoided, although under faster growing conditions (Apr-Oct inclusive) it might be appropriate to increase this to 1.5g/l.

3.2.2 Investigation into the effects on the growth of tomato in the BSI PAS 100:2011 growth test as a result of varying rates of dilution of composted green waste samples with a range of EC levels by peat

Four compost samples with EC values ranging from 600 to 2000 µS/cm were diluted with peat at a range of rates depending on their EC, namely, 1:1 (50% peat), 2:1 (67% peat), 3:1 (75% peat), 4:1 (80% peat) and 6:1 (86% peat) to determine the effect on the emergence and growth of tomato. The compost samples were supplied from commercial companies or were blended using material from several sources to produce four samples with the required target EC levels (600, 900, 1500 and 2000μS/cm). These blends were compared to the standard PBGM with KCl added at 0, 0.5, 1.0, 1.5 and 2g/l of peat to provide a range of EC levels as controls. All the growing media were mixed on 15 December 2010 with tomatoes sown one day later. Results: Measured EC values for the various peat:CGW test mixes and the PBGM controls with 0-2g/l KCl added are given in Table 3-7, where it can be seen that the mixes tested had final EC values from 300 to just over 1000μS/cm.

Table 3-7 EC values for all composts and PBGM media employed

Dilution ratio peat:CGW

Compost (original EC) Peat %

A (698μS/cm)

B (980μS/cm)

C (1695μS/cm)

D (2037μS/cm)

1:1 710

50

2:1 579

67

3:1 574 622 776 1045 75

4:1

680 780 80

6:1

526 649 86

KCl rate Peat only mixes (peat EC 29μS/cm) Peat %

0.0g/l 300 100

0.5g/l 476 100

1.0g/l 654 100

1.5g/l 810 100

2.0g/l 1020 100

Seedling emergence was earliest for the peat treatments, but with a significant delay where KCl had been added at a rate above 1g/l of peat. For the peat and composted green waste mixes seedling emergence was delayed for the lower dilutions and for the materials with high EC. However, by 12 days after sowing, emergence was similar for all treatments, although it was still lower where KCl had been added at 1.5 and 2g/l to the PBGM. Final emergence ranged between 93 and 100%.


Data for the assessment 10 days after sowing are shown graphically in Figure 3-3 and show the clear negative dose response to KCl addition (and increasing EC), whilst the responses to dilution (and therefore, reduction of EC) for the various compost samples were less regular, especially with composts C and D.

Figure 3-3 Percentage tomato seedling emergence versus peat content of growing media in a BSI PAS 100:2011 growth test after 10 days

Figure 3-4 Tomato plant harvest weight (g) versus peat content of growing media in a BSI PAS 100:2011 growth test

Plant size was initially larger for plants growing in peat mixes (PBGM) and peat plus the two CGW materials with the lowest EC. By 21 days after sowing plant size was smaller for media

0

10

20

30

40

50

60

70

80

90

100

100 86 80 75 67 50

% e

me

rge

nc

e

% peat in growing medium

PBGM (Nil KCl)

PBGM (0.5g/l KCl)

PBGM (1.0g/l KCl)

PBGM (1.5g/l KCl)

PBGM (2.0g/l KCl)

CGW A

CGW B

CGW C

CGW D

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

100 86 80 75 67 50

g/p

lan

t

% peat in growing medium

PBGM (Nil KCl)

PBGM (0.5g/l KCl)

PBGM (1.0g/l KCl)

PBGM (1.5g/l KCl)

PBGM (2.0g/l KCl)

CGW A

CGW B

CGW C

CGW D


where the EC was above 700μS/cm (peat + CGW A @ 1:1, peat + CGW D @ 1:3 and peat + 2g/l KCl). Plant colour remained similar until 21 days when the peat only treatments started to develop mottled foliage, although this became less marked in the few days before harvest. As can be seen in Figure 3-4, mean plant weights at harvest (32 days after sowing) were similar for all peat + composted green waste mixes. Mean plant weight was only significantly (and critically more than 20%) lower than the PBGM (zero KCl addition) in the peat + CGW D @ 3:1 mix and where potassium chloride had been added to the peat controls at above 1g/l. However, this peat + CGW mix was only 14% lower than the peat + 1g/l KCl control and thus would have met the BSI PAS 100 pass rate criterion if this had been used as the basis of the test. Conclusions:

Whilst increasing dilution rates for the high EC composted green waste materials above the current maximum ratio (4:1) would minimise the effects of EC or other compost factors, the proportion of material in the mix would be too low to properly determine phytotoxic risks from the compost sample;

In order to accurately and fairly assess the safety of high EC composts to plants without excessive dilution, it would be desirable to compare its performance with a peat-based control (PBGM) augmented by the addition of potassium chloride to increase the EC; probably at a rate of 1g/l KCl; and

A single dilution rate of 3:1 (75% peat + 25% CGW) for all test materials (with an EC of up to 2000μS/cm) and comparison with two peat-based controls (0 g/l and 1g/l KCl added) is worthy of consideration.

3.2.3 Sunflower A late, small-scale trial was conducted to determine the salt sensitivity of this species. Sunflower seeds (cv Valentine) were direct-sown into PBGM and also mixes of peat plus compost which had an EC level of either 1000µS/cm or 2000µS/cm. The peat and composts were mixed at 3:1 on 23 February 2011 with seeds sown the same day. The test was set up in the glasshouse with standard recording and assessments carried out after emergence. There were three replicates of each of the three treatments and the trial ran for 28 days. It was found that the speed of emergence and the total number of seedlings which emerged was unaffected by treatment, and that foliage colour and plant quality was similar for all treatments. 28 days after sowing the plant weights were also similar, with the mean values for the two compost treatments being 94% and 97% of the PBGM control (7.08g per plant). However, in one of the PBGM replicates only 8/10 seeds emerged, and this led to a markedly higher individual plant weight; at least 18% higher than any other replicate. Thus, whilst the results indicate that salt tolerance is unlikely to be a significant issue with this species, they do suggest that results might not be as uniform and robust as with tomato and field bean. This aspect was investigated further in a validation trial. 3.3 Validation trial This trial was the culmination of the experimental programme, and brought together for the first time in one experiment: herbicide bioassay, composts with a range of ECs, and PBGM controls with and without KCl addition in a partial factorial experiment. The objective was to confirm the robustness and practicability of the application of the preliminary findings (reported in Sections 3.1 and 3.2 above) to the BSI PAS 100:2011 plant response test.


3.3.1 Species 1. Tomato (Lycopersicon esculentum) cv. Shirley (current requirement in BSI PAS 100:2011

plant response test); 2. Field bean (Vicia faba) cv. Fuego (best overall alternative species from preliminary

studies); and 3. Sunflower (Helianthus annuus) cv. Valentine (alternative species representing

Asteraceae). For practical and operational reasons these species were tested concurrently in three separately-run trials of the same design on adjacent benches of the same glasshouse, under the same conditions. All crops were sown on 11 March 2011 and harvested on 9 April 2011. 3.3.2 Herbicide rates To simulate contamination of compost with clopyralid, six rates of herbicide from 0 – 0.333mg ai/l13 were carefully applied to each of three compost blends prior to dilution with peat. These are set out below in Table 3-8.

Table 3-8 Rates of clopyralid applied to compost blends in the Validation Trial together with their relationship to those used in screening experiments

Rate Code

Clopyralid rate applied to CGW (mg ai/l)

Clopyralid rate applied to CGW (ppb or ppb)

Concentration of clopyralid

solution applied to

peat:CGW substrate in

screening trials providing

same effective dose rate

0 Nil - water only control 0 0

I 0.0000333 33ppt 250ppt

II 0.000333 333ppt 2.5ppb

III 0.00333 3ppb 25ppb

IV 0.0333 33ppb 250ppb

V 0.333 0.333ppm or 333ppb 2.5ppm

As can be seen from Table 3-8, the rates chosen match those rates in the screening trials that, at the upper levels, produced clear symptoms in field bean and even tomato. These rates can also be seen to cover the theoretical ‘realistic worst case’ and ‘low’ levels of clopyralid residue calculated in Appendix 3, of 0.14 and 0.00015 mg ai/l respectively. The lower rates were included to investigate whether the bioassay species would exhibit greater sensitivity as seed-sown plants rather than as seedlings treated post-emergence, and as glasshouse growing conditions improved with increased levels of solar radiation in the spring.

13 ai = active ingredient


3.3.3 Growing media Three peat-only control media (PBGM) with and without added KCl plus three composts with different ECs (each blended 3:1 with peat) were employed as shown below.

Peat only control (PBGM);

Peat only control (PBGM) + 1g KCl/l of peat;

Peat only control (PBGM) + 1.5g KCl/l of peat;

Peat + CGWA* with a measured EC of 673uS/cm (3:1 mix);

Peat + CGWB* with a measured EC of 1300uS/cm (3:1 mix); and

Peat + CGWC* with a measured EC of a 2000uS/cm (3:1 mix).

*These are each composite blends of test samples from different sources to provide CGWs with distinct ECs for testing: all were assumed to be herbicide-free. 3.3.4 Experimental design For each species the experiment was a partial factorial design as follows, with clopyralid applied only to growing media treatments 4-6 above: ((6 herbicide rates x 3 Peat:CGW mixes) + 3 x PBGM) x 3 replicates = 63 trays 3.3.5 Method The experiment was carried out in accordance with the standard BSI PAS 100 procedure. However, a number of precautions were employed to minimise the risk of cross contamination:

New mixing implements were used for each herbicide treatment;

Mixing was carried out on new polythene sheets for each treatment and afterwards placed in new polythene bags;

New wooden press boards to make indentations for the seeds were used for each herbicide treatment and discarded after use;

Trays with nil herbicide incorporation were filled first;

Seed sowing started with the untreated controls and then sequentially through the herbicide treatments beginning with the lowest rate;

Each treatment was completed (including covering of the seed) before moving onto the next treatment;

The trial was carried out on a mesh bench covered with polythene and capillary matting to capture run-off. Each half seed tray was stood on an upturned half seed tray to isolate it from the capillary matting;

The herbicide treatments were arranged in discrete randomised blocks in rate order down the bench to reduce the potential risk of cross contamination by vapour or water splash during overhead watering14. All non-herbicide treated controls were similarly randomised in a separate block farthest from the highest herbicide rate15;

Each block of treatments was separated from the next block by an unsown but watered tray of compost;

Extra trays were sown and used as guard trays to reduce potential end edge effects. At the edge of the trial next to the highest herbicide levels these guards provided an additional check on herbicide transfer or drift; and

At the end of the trial all materials were treated as contaminated waste and disposed of appropriately to avoid long-term contamination at STC.

14 This was a precautionary approach as clopyralid is not known for having high levels of vapour activity 15 Note: Experience indicated that variation along the length of the bench was not a significant factor in plant performance.


3.3.6 Assessments The following routine assessments were undertaken:

Emergence counts commencing when the seedlings reached the expanded cotyledon stage 6-29 days after sowing (according to crop);

Plant size 14, 21 and 29 days after sowing (1-10 scale);

Records of herbicide damage 14, 21 and 29 days after sowing (0-5 scale based on stem and leaf distortion);

Records of any stress symptoms associated with high ECs in the growing media (based on emergence rate and final plant stand);

Plant weight at 29 days after sowing;

Determination of relative crop variability (CV%) for key metric of mean plant weight at harvest; and

Photographs of treatment effects.

3.3.7 Results and discussion Seedling emergence The rate of tomato seedling emergence at eight days was fastest in the standard PBGM. It was markedly slower (at eight days) where potassium chloride had been added at 1.5g/l of peat. For all other treatments the rate of emergence was similar. By 14 days all treatments had achieved 93-100% germination. At 29 days there had been no further emergence and no tomato plant death. There were no significant differences within peat + CGW combinations in the absence of herbicide. In field bean, seedling emergence in the non-herbicide treated media was much slower where potassium chloride had been added at 1.5g/l of peat. However, in this crop, emergence was also influenced by herbicide inclusion and was very markedly retarded in the three media with the highest rate of clopyralid, where many plants failed to produce their first set of leaves. The uniformity of emergence of this crop appeared more variable than for tomato but this may have been due to its greater susceptibility to clopyralid. By 29 days after sowing there had been some field bean plant death at the highest rate of herbicide incorporation. In sunflower, the rate of seedling emergence was similar for all treatments but was delayed where potassium chloride had been added at 1.5g/l of peat and at the highest dose of clopyralid. Maximum emergence was achieved in all treatments by 14 days but by 29 days after sowing there had been significant sunflower plant death at the highest rate of clopyralid. Data for all three crops at the critical 14-day mark are shown in Figure 3-5for all the peat:CGW mixes at six rates of clopyralid inclusion and show that clopyralid had a significant effect on this metric in all crops – but only at the highest dose rate.


Figure 3-5 Percentage of seeds sown that produced seedlings with expanded leaves 14 days after sowing for tomato, field bean and sunflower in peat + CGWs treated with clopyralid

0

20

40

60

80

100

0 I II III IV V

Peat + CGW C Peat + CGW B Peat + CGW A

0

20

40

60

80

100

0 I II III IV V


0

20

40

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0 I II III IV V


Tomato Field bean

Sunflower

Rate code Clopyralid rate (mg ai/l CGW)

0 Nil

I 0.0000333

II 0.000333

III 0.00333

IV 0.0333

V 0.333

Note: EC of CGWs in µS/cm; A = 673, B = 1300, C = 2000

The assessments made at 14 days show that in none of the three crops was there any interaction between CGW conductivity and clopyralid dose rate. Plant size Tomato plants in most treatments were of similar size except at the highest rate of clopyralid, where at 14 DAS they were less than half as big and by 21 DAS only one tenth of the size of all other treatments. The main reason for this was the failure of the seedlings to develop beyond the expanded cotyledon stage. Field bean plant size was severely reduced at the highest rate of clopyralid and to a lesser extent at the second highest rate (0.0333mg ai/l). By 29 days after sowing all the CGW treatments were smaller than the peat only controls. Sunflower plant size was severely affected at the highest rate of clopyralid and to a lesser extent at the second highest rate (0.0333mg ai/l). Overall there did not appear to be any consistent effect of EC level in the CGW on plant size in any crop. However, in the case of tomato and field bean at 14 and 21 days after sowing, the plants growing in the PBGM with potassium chloride added at 1.5g/l of peat were smaller. Herbicide damage observations In tomato, damage was only recorded at the two highest herbicide rates. This ranged from severe distortion of the growing point and failure to develop properly after the cotyledon stage at 0.333mg ai/l of CGW to slight distortion of the leaves in the plant head at 0.0333mg ai/l of CGW. At rates of 0.00333 mg ai/l of CGW or less no damage was observed, confirming its relatively low level of sensitivity to clopyralid. In field bean, damage ranged from severe distortion of the growing point and failure to develop properly after emergence at the highest rate of clopyralid inclusion (0.333mg ai/l of CGW) to slight distortion of the leaves at the lowest level of inclusion, 0.0000333mg ai/l .


There was also diagnostic herbicide damage on plants in the peat plus CGW controls to which no clopyralid had been experimentally added.

Figure 3-6 Herbicide damage level (0-5) 29 days after sowing for tomato, field bean and sunflower in peat + CGWs treated with clopyralid

0

1

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Tomato Field bean

SunflowerRate code Clopyralid rate (mg ai/l CGW)

0 Nil

I 0.0000333

II 0.000333

III 0.00333

IV 0.0333

V 0.333


Herbicide damage in sunflower ranged from severe distortion of the growing point and failure to develop properly after the cotyledon stage at the highest rate of inclusion (0.333mg ai/l of CGW) to slight distortion of the leaves in the plant head at 0.00333mg ai/l of CGW. At the 0.0333 and 0.00333 mg ai/l of CGW the symptoms were not present on all the plants in the tray. At a rate of 0.000333mg ai/l of CGW and below, damage symptoms were absent except in the peat + CWG A treatment. Data for all three crops immediately prior to harvest after 29 days for all the peat:CGW mixes at six rates of clopyralid addition are presented in Figure 3-6. These show graphically that field bean was the most sensitive crop to the presence of clopyralid, followed by sunflower and then tomato. Furthermore, field bean was the only species to produce diagnostic symptoms of herbicide damage across the full spectrum of clopyralid rates evaluated in all experimental mixes. Field bean also exhibited specific diagnostic symptoms in CGW mixes that had not been treated with clopyralid, which indicates that there was probably a (hitherto undetected) low level of herbicide contamination in some of the CGW material used here for experimental purposes. This is not considered to be a ‘false positive’ result nor due to cross contamination, but a genuine indication that hormone herbicide residues were present in some of the CGW samples used to prepare the experimental blends – especially CGW A. Such symptoms are not consistent with the effect of factors such as the presence of volatile organic acids or other substances present in inadequately composted material, because


these tend to produce root damage and attendant weak growth rather than distortions of the aerial portions of the plant. Plant weight at harvest Plant weights were lowest at the highest rate of clopyralid due to the severe effects of the herbicide on the growth and development of all three crops. For field bean, mean plant weights were inversely related to the clopyralid dose rate applied to the CGW across the full range of applied concentrations. For sunflower, mean plant weights were reduced at the two highest rates but the response was not consistent, and tomato was negatively affected only at the highest clopyralid level. Data for all three crops at harvest for all the peat:CGW mixes at six rates of clopyralid addition are presented in Figure 3-7. This graphic shows clearly that, of the three crops tested, field bean exhibited the most uniform and consistent inverse dose response to clopyralid. There was surprisingly little response to increasing EC level in the CGW up to 2000μS/cm in any of the crops under test. This is a different result from that obtained in the preliminary study with tomato begun in December 2010 (cf. Figure 3-4) and probably indicates a seasonal influence - consistent with greater plant tolerance of salt levels under better growing conditions and when irrigation is increased.

Figure 3-7 Mean plant harvest weight (g) for tomato, field bean and sunflower in peat + CGWs treated with clopyralid

0

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8

0 I II III IV V


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Tomato Field bean

Sunflower

Rate code Clopyralid rate (mg ai/l

CGW)

0 Nil

I 0.0000333

II 0.000333

III 0.00333

IV 0.0333

V 0.333



Crop variability From visual assessments of individual plants when harvesting trays, field bean plants appeared to be more variable in size than tomato in this experiment. Since individual plant weights were not recorded, this effect cannot be quantified. However, to examine the possible effect of this (at the tray level), the variability of the mean plant weights across the three replicates contributing to the overall mean for each treatment in each crop has been calculated and these data (Coefficients of Variation) are presented alongside each mean in Table 3-9.

Table 3-9 Mean plant harvest weights (g) for tomato, field bean and sunflower in all treatments together with calculated CV% for each

Growing media and herbicide treatment (mg ai/l CGW)

Tomato Field bean Sunflower

Mean CV(%)a Mean CV(%)a Mean CV(%)a

Peat (PBGM) 4.46 20 4.80 8 5.61 6

Peat + KCl @1g/l 4.82 22 6.05 18 6.49 9

Peat + KCl @1.5g/l 4.39 16 5.81 12 5.67 16

Peat + CGW A 5.17 13 5.07 8 5.83 1

Peat + CGW B 5.12 7 4.92 17 5.66 15

Peat + CGW C 5.78 25 4.96 7 5.65 23

Peat + CGW A

Clopyralid 0.333 0.49 51 0.5 25 0.3 34

Clopyralid 0.0333 5.74 22 2.37 22 3.04 30

Clopyralid 0.00333 6.67 14 3.28 32 5.64 17

Clopyralid 0.000333 6.38 24 3.92 6 4.87 15

Clopyralid 0.0000333 5.54 15 4.43 13 4.26 4

Peat + CGW B

Clopyralid 0.333 0.83 53 0.56 15 0.36 41

Clopyralid 0.0333 6.56 8 2.36 11 3.57 5

Clopyralid 0.00333 6.19 23 4.31 8 5.55 25

Clopyralid 0.000333 6.06 16 4.59 8 4.51 20

Clopyralid 0.0000333 5.7 18 4.51 8 4.75 2

Peat + CGW C

Clopyralid 0.333 0.52 44 0.45 62 0.32 24

Clopyralid 0.0333 5.61 30 2.84 11 2.5 25

Clopyralid 0.00333 6.09 7 3.58 9 4.9 11

Clopyralid 0.000333 4.72 7 4.48 19 4.11 13

Clopyralid 0.0000333 4.93 22 4.06 8 4.47 17

Mean CV(%)

22

16

17

Mean CV Ranking

2.29

1.81

1.81 a Coefficient of variation (Standard Deviation of the mean expressed as a % of the mean value)

This analysis shows that, as in the earlier study (cf. Table 3-5), overall mean plant weight – at the tray level - was a less variable metric in both field bean (cv. Fuego) and sunflower than in tomato (cv. Shirley). Field bean ranked first for the lowest CV% (i.e. lowest crop variability) in nine of the twenty-one treatments and ranked third in five. By comparison, tomato ranked first only five times but ranked third eleven times.


Therefore, using Vicia faba cv. Fuego as a bioassay species at 10 seeds per tray would be potentially more robust than using Lycopersicon esculentum cv. Shirley at the same planting density. Performance relative to controls Two metrics are used to judge the quality of CGW samples in the BSI PAS 100 tomato growth test. To ‘pass’, samples must achieve at least 80% of the PBGM control result for both seedling emergence at 14 days and mean plant weight at 28 days after sowing. In this trial, treatments in all three crops (other than in the highest clopyralid rate with field bean) achieved 87 -117% of the seedling emergence in their three peat-based growing media controls with or without KCl addition. Therefore with the exception specified, all CGW samples would have ‘passed’. With plant weight the situation is not as straightforward as can be seen from Figure 3-8.

Figure 3-8 Mean plant harvest weight in tomato, field bean and sunflower for mixes of peat + CGWs treated with clopyralid relative to three different peat-based growing media controls – treatments expressed as a percentage of control (29 days after sowing)


In all three crops, plant growth and harvest weight was dramatically reduced at the highest rate of clopyralid addition and these treatments all ‘failed’ – just as with seedling emergence. Setting aside the highest rate of clopyralid incorporation, it can be seen that in tomato, plant weight relative to all controls increased slightly with increasing dose of clopyralid up to 0.0333mg ai/l CGW, and the presence of the herbicide had no effect on the pass rate. In field bean the choice of control had a marked effect on the number of treatments ‘passing’. Indeed, except at the zero herbicide dose rate, all treatments ‘failed’ where 1.0g/l or more KCl was added to the PBGM and this suggests that, were field bean to be adopted as the bioassay species of choice, KCl augmentation of the controls would be unnecessary and undesirable. This is because in this crop the addition of KCl to the controls provided a more significant boost to growth than in tomato. However, it is interesting to note that in this crop, plant weight provided direct quantification of adverse herbicide concentration effects - independently of subjective assessments of symptoms. In sunflower, there was a less clear-cut position, with many herbicide-dosed treatments failing to beat the 80% threshold even when compared with the PBGM control without any supplementary KCl addition. 3.3.8 Conclusions

Seedling emergence and final plant number was generally unaffected by the EC level in the compost, but there was slower emergence where potassium chloride had been added at 1.5g/l of peat. This confirms previous findings where high EC reduced the speed of emergence but not final plant stand;

Seedling emergence was largely unaffected by the rate of clopyralid, but field beans failed to reach the expanded first leaf stage at the highest rate of clopyralid used in this study (0.333mg ai/l CGW); if this species is to be used in the standard growth test then a different growth stage description would have to be used, possibly ‘seedling emerged and shoot visible’;

Except at the two highest rates of incorporation, tomato exhibited no significant adverse responses to clopyralid;

Field bean was the most sensitive species to clopyralid, and was the only species to express diagnostic herbicidal symptoms at the lowest rate of incorporation. This crop also exhibited an inverse dose-related response to clopyralid concentration in respect of plant weight at harvest, which is potentially useful as it provides objective data to support the subjective assessments of damage symptoms;

Sunflower did not exhibit as high a level of sensitivity to clopyralid as expected in this test and where symptoms were expressed they were not particularly diagnostic and did not affect all the plants in the tray;

There did not appear to be any enhanced herbicide symptom expression or plant growth reduction associated with increasing CGW EC level per se - up to 2000µS/cm;

No herbicide damage was observed in the peat only controls, but hormone damage was exhibited in field bean in all three untreated peat plus composted green waste mixes indicating that some of the composted green waste material used to produce these blends probably had a very low level of synthetic auxin herbicide contamination. This was more severe for the blend with an EC of 673µS/cm (CGW A);

The absence of any herbicide damage on the peat only controls and also on the guard trays adjacent to the highest rate confirmed that that there had been no cross-contamination and that it may be possible to undertake the test in the same area of the glasshouse16. In any case, the use of capillary matting under the trays in the current test

16However, this will depend on the type of herbicide contamination as the phenoxy-carboxylic group herbicides such as MCPA are more active in the vapour phase than clopyralid and

aminopyralid i.e. the pyridine-carboxylic group – especially at higher glasshouse temperatures.


method will have to be avoided in future to prevent any risk of leachate contamination from adjacent trays;

There is a very strong case for the use of field bean as a bioassay species within BSI PAS100. Initially it should be used as the supplementary indicator species for potential herbicide contamination in compost samples; especially those destined for sensitive applications. It could either be used as a simple observational crop or used in a fully replicated test (in parallel with tomato) to provide a useful quantitative metric. However, until more experience is gained on the performance of this species under a wider range of environmental conditions and with uncontaminated CGW samples that may have other phytotoxic components, tomato should be retained;

In the present state of knowledge (and other than as an indicator of clopyralid contamination) tomato remains the most reliable species to assess the general suitability of composts for growing plants - for a wide range of salt levels;

More work is required to determine under which circumstances and for which species KCl should be added to PBGM controls as this may have a complicated effect on the number of compost samples meeting the minimum requirement of 80% of the mean plant weight in the control; and

The choice of control appears to have little effect on the achievement of 80% seedling emergence in the control at 14 days.

4.0 Discussion It is widely recognised that synthetic auxin hormone weedkillers used to treat lawns, grassland or agricultural and horticultural crops can enter the green waste stream and that some are incompletely broken down during the composting process. These herbicides and their breakdown products can be phytotoxic to some plant species at levels below 10ppb and thus render the compost output potentially ‘unfit for purpose’ if it is to be used in sensitive applications. Low levels of contamination are often below the level of detection by chemical analysis in the laboratory and instead a plant bioassay is required for effective detection. 4.1 What are sensitive crops and applications? At least three large and important plant families are known to be highly sensitive to synthetic auxin herbicides including clopyralid. These are: Asteraceae (Compositae), Fabaceae (Leguminoseae) and Solanaceae. Because of their sensitivity, representatives of all three have been evaluated here as candidates for a combined herbicide bioassay and growth test; namely, dahlia, sunflower, field bean, lentil and pea; plus tomato (current BSI PAS 100:2011 growth test species). These families include some important edible and ornamental horticultural crops (especially bedding plants), although it should be noted that sensitive ornamental species are not limited to these three families and that many other important genera are known to be sensitive, such as Geranium and Pelargonium (Family Geraniaceae) and Lobelia (Family Campanulaceae). Indeed the complex of species in the ornamentals sector is such that it is prudent to assume that they are all potentially sensitive. It is obvious that minimal risks from herbicide residues are posed to crops on which the herbicide(s) were originally recommended for use - generally monocotyledonous species including, in the case of clopyralid, grass, cereals, maize and onions. Members of the Brassica family can also be treated and are considered to be at low risk from clopyralid (although they may be sensitive to other synthetic auxin herbicide contaminants). Established crops such as orchard and cane fruit, and shrubs which are only exposed to compost via mulching are unlikely to be at significant risk either.


Although it is possible to qualitatively identify synthetic auxin herbicide contamination through bioassay, it is not possible to use this method to identify which synthetic-auxin herbicide is present. As different synthetic auxin herbicides operate in different ways and break down at different rates, it is less easy to be certain about the fate of their residues in the soil and, whilst applications of contaminated compost may be made to ‘safe’ crops in one season, they could have an adverse effect on a subsequent sensitive crop17. Even if applied to permanent grassland (or a ryegrass/clover ley), contaminated compost might detrimentally impact on clover in the sward18. 4.2 What is the best bioassay species to use? Our studies have shown that field bean (Vicia faba) would be the best herbicide-responsive species to use in the BSI PAS100:2011 growth test19. There are many reasons for this:

It is highly sensitive to clopyralid and several other key hormone weedkillers at low (but realistic) levels of potential contamination that would be problematic in sensitive crops;

It exhibits clear diagnostic symptoms even at low levels of contamination;

In the validation trial, harvest weight reduced progressively as the clopyralid dose rate increased;

It has a large seed that is easy to work with, has an upright habit and grows quickly to produce robust plants - even under poor light conditions;

Plant mass per tray is more consistent than tomato (although there may be more variability between individual plants), but this could potentially be improved with better seed selection; and

Contrary to literature indications quoted in Table 3-2, it was tolerant of high ECs in the compost (up to ca. 2000μS/cm, the maximum used in the trials).

Nevertheless, we currently have insufficient experience with field bean under a full spectrum of growing conditions, in test situations other than a glasshouse or with a wide range of composts. We do not know how this species responds when exposed to compost that may be immature or that contains phytotoxic factors other than hormone weedkiller residues (such as volatile organic acids). Therefore, whilst field bean is a very strong candidate to replace tomato in the BSI PAS 100 growth test, it would be advisable to collect more performance data on this species - growing it alongside tomato for an interim period at the very least. This work should be carried out at a competent testing facility with the necessary expertise. It is recognised that this will increase testing costs, but there may be few options in applications where the risk of damage from herbicide contaminated compost is perceived to be high and confidence in the bioassay is vital. Tomato remains the test species for which we have most confidence as a general indicator of ‘fitness for purpose’ even though it was shown to be relatively insensitive to clopyralid. 4.3 Composts with high EC Assessing the best course of action for dealing with compost with a high EC is problematic, as observations made during the validation trial have been at variance with those from the preliminary studies. This appears to be because of the pronounced difference in growing conditions for the two phases of work. The initial studies were conducted under the worst possible winter glasshouse conditions of low ambient light and freezing outside temperatures

17 As confirmed in Section 8.2 of the report by Gilbert et al. (2009). However, Andrew Bailey (Principal Biologist, Dow AgroSciences – pers comm) considers this a ‘most

unlikely scenario’. 18 Andrew Bailey (Principal Biologist, Dow AgroSciences – pers comm) has commented that established clover in a grass ley is much more tolerant than clover grown in

pots; although damaged it often survives a field application of clopyralid at 200gai/ha. 19 This is the same bioassay species recommended to gardeners by Dow AgroSciences to check for aminopyralid residues in manure. Andrew Bailey (pers comm) has commented that this has now been used for about two years and that ‘it seems to be very robust’.


(notwithstanding the provision of heat and artificial light as specified for the growth test procedure), whilst the validation trial was conducted under favourable spring growing conditions. This meant that plants grown in phase one were much more stressed by high salt levels in the CGW than those in phase two, and responded differently. The preliminary study showed that augmenting the PBGM control with KCl (at 1.0 or perhaps even 1.5g/l peat) could make it a more appropriate comparison with high EC compost diluted at a ratio of 3:1 with peat. This was because it increased the EC of the PBGM to levels in the peat:CGW mixes and thereby reduced the performance differential between the test samples and the controls. However, in the validation experiment, augmenting the controls with KCl at both 1.0 and 1.5g/l (whilst still reducing the rate of germination) did not significantly reduce mean tomato plant weights, whilst mean weight of field bean plants were significantly increased. Another indication of lower sensitivity to EC under spring growing conditions was that mean plant weights for all three crops in the unspiked 3:1 peat:CGW mixes were very similar irrespective of the original EC of the CGW used. It seems that in tomato, a standard 3:1 ratio can be justified for all CGW ECs (up to 2000μS/cm and maybe higher) - without KCl augmentation of the control - especially in good growing conditions. In winter (or other times of low solar radiation in autumn or early spring) augmentation of the PBGM with 1.0g/l KCl would seem to provide a more appropriate control for this crop. In field bean and sunflower however, KCl augmentation of the PBGM control would appear to be unnecessary, certainly under good growing conditions, as it enhances the growth of these crops in the PBGM and puts the test material at a disadvantage. 5.0 Overall conclusions & recommendations 5.1 Objective 1 ‘Ensure’ that the BSI PAS 100:2011 plant response test is sufficiently sensitive to detect traces of persistent herbicides in compost (including aminopyralid, clopyralid and triclopyr20) at concentrations that could prove damaging to sensitive plants when the composts are used in different markets (for example: use as a soil improver in agriculture and field horticulture or use in growing media) 1. The use of tomato in the BSI PAS 100:2011 plant growth test does not achieve the

objective of ensuring the detection of clopyralid in compost at concentrations that could prove damaging to sensitive plants when the composts are used in different markets. Nevertheless, for the time being, tomato should be retained as the species of choice to provide a general indication of ‘fitness for purpose’.

2. For the detection of clopyralid and some other synthetic auxin herbicide residues at critical low concentrations in composted green waste, field bean (Vicia faba cv. Fuego) should be used as the bioassay species. However, since our knowledge of the growth response with field bean to a wider range of composts and test situations is still limited, it should initially be used alongside tomato as a qualitative observational crop (possibly just a single tray or pot to limit costs).

3. To facilitate the testing and use of field bean as a qualitative synthetic auxin herbicide detector, a protocol has been developed by the research team as a standalone document that includes photographs of diagnostic symptoms and guidance notes.

4. Species in the following key plant families21 are sensitive to clopyralid and/or aminopyralid (and other synthetic auxin herbicides):

a. Asteraceae or Compositae (daisy family)

20 Triclopyr was not evaluated experimentally as the desk study established that this herbicide was rapidly degraded during composting. 21 Whilst these are the principal plant families there are very many genera in other families (particularly ones employed in ornamental horticulture) which are also sensitive

to synthetic auxin herbicides, such as Geranium and Pelargonium (Family Geraniaceae) and Lobelia (Family Campanulaceae). Indeed the complex of species in the ornamentals sector is such that it is prudent to assume that they are all potentially sensitive.


b. Chenopodiaceae (beet family) c. Fabaceae or Leguminosae (pea and bean family) d. Solanaceae (potato and tomato family) e. Umbelliferae (carrot family)

5. Compost intended for soil incorporation or growing media production in connection with such species (examples of which are given in Appendix 2) should be subjected to a bioassay test with field bean.

6. Care must be exercised in the following applications to avoid the use of composts contaminated with synthetic auxin herbicide residues:

a. In all protected and ornamental horticulture applications - as a very large range of highly sensitive plants (within and without the principal families listed above) are grown in soil and growing media in which compost is incorporated.

b. In clover leys or swards where clover is a valued constituent - as this leguminous plant could be adversely impacted.

c. In rotational cropping situations where a sensitive crop will follow a tolerant one - since the quantity and persistence of any residues and their effect on subsequent cropping cannot be estimated22.

7. For the bioassay to be meaningful it is essential that samples of compost submitted for testing shall be truly representative of the batch. Composters should be reminded that it is therefore essential that they follow the sampling protocol set out in AfOR’s ‘Guidelines for Compost Sampling’23 which accords with BS EN 12579 ‘Soil improvers and growing media - Sampling’.

5.2 Objective 2 ‘Ensure’ that this test remains fit for purpose; delivering the required sensitivity to herbicide residues whilst accommodating composts with higher than average salt contents (such as those partially derived from food wastes) – ideally in a single test. It is not possible to make definitive recommendations to accommodate compost with high EC in the BSI PAS 100 test, as the results were confused by seasonal factors. Nevertheless, the following points need to be considered: 1. The ratio of peat:CGW should ideally be standardised as, under the current procedure,

low EC compost receive less dilution (of phytotoxic factors other than salts) whilst those with high EC receive more. Furthermore, the variability of the nutrient contribution from the compost in the test mixes is skewed by differential dilution of the samples.

2. Dilution of the test material beyond the current limits of four parts peat to one part CGW (i.e. just 20% CGW by volume) cannot be recommended without compromising the value of the test entirely.

3. For tomato: a. The peat to CGW ratio could be fixed at 3:1; but b. KCl augmentation of the PBGM (at 1.0g/l) may be required under poorer

growing conditions in glasshouses during winter and in early spring or late autumn24.

4. For field bean: a. The peat to CGW ratio could be fixed at 3:1; but b. KCl augmentation the PBGM appears to boost rather than diminish the control

and is, at least under spring glasshouse conditions, unnecessary and undesirable.

22 Residues of some herbicides (e.g. picloram) can persist for over a year but the risk will be dependent on the initial herbicide concentration, the rate at which the CGW was applied and the sensitivity of the following crops. 23 Document 32 at http://www.organics-recycling.org.uk/page.php?article=1894&name=AfOR%27s+templates+aligned+to+PAS+100%3A2011 24 At these times, despite using artificial light and heating as prescribed in the protocol, sunlight and solar (temperature) gain is usually low, plant growth is slow and irrigation is restricted; so the effects of salt stress in the test mixes is most apparent.

http://www.organics-recycling.org.uk/page.php?article=1894&name=AfOR%27s+templates+aligned+to+PAS+100%3A2011


6.0 Further work 1. Field bean should be developed as a single test species for both quantitative and

qualitative aspects of the BSI PAS 100 plant response test – to replace tomato. To facilitate this, more experience is required on the use of this species with routine compost samples in replicated tests to assess its behaviour in different composts, in different test conditions and at different seasons – and of course to assess how easily operators are able to spot diagnostic symptoms of herbicide contamination using reference photographs. This should be undertaken as a project independent of the observational testing referred to above.

2. The response of tomato and field bean to composts of known levels of maturity and volatile organic acid content has not been critically assessed before. This should be undertaken under a broad range of growing conditions and could be combined with a second study into the best approach to use with composts of high EC (including limited dilution with peat no greater than 4:1 and the use of KCl augmentation to the PBGM control).

3. Only one cultivar of field bean has been used in this study. Since not all cultivars will respond similarly, consideration could be given to the screening of others as these might prove more sensitive, more expressive, faster growing, more tolerant of poor growing conditions and/or more uniform in growth and response.

4. If it is acceptable to retain tomato as the main crop and to run a herbicide-sensitive crop alongside it for qualitative detection of herbicide residues only, further consideration might be given to the evaluation of the use of clover as the bioassay species as this species might be able to give test results more quickly and more cheaply.

7.0 Photography Close-up photographs of diagnostic hormone herbicide damage symptoms in tomato, field bean and dahlia from the preliminary studies and of clopyralid damage symptoms in trays of tomato, field bean and sunflower from the validation trial are presented in Appendices 10 to 15.


Appendix 1

Extracts from Dow AgroSciences’ UK product labels for pyridine-carboxylic acid herbicides Active Ingredient

Common Name

Dow Product

Name On-label uses Important crop sensitivity information

Product Persistence and Degradation (Based largely on data

for active ingredient)

Aminopyralid Forefront* (combined with

fluroxypyr). Also Pharaoh*

(combined with

triclopyr)

Grassland for grazing only FOREFRONT residues in plant tissues which have not completely decayed may affect

succeeding susceptible crops e.g. peas beans and other legumes, sugar beet,

fodder beet, carrots and umbelliferae,

potatoes and tomatoes, lettuce and other compositae. Do not plant potatoes, sugar

beet, fodder beet, vegetables, beans or other leguminous crops in the next

calendar year following application. Do not plant potatoes, sugar beet, fodder beet,

vegetables, beans or other leguminous

crops in the next calendar year following an application of manure from animals fed

on grass treated with FOREFRONT or fodder resulting from grass treated with

FOREFRONT. Under no circumstances

should manure be supplied to gardeners or allotment holders, or commercial compost

producers; i.e. there must be no off-farm sale or supply. Do not use any plant

material treated with FOREFRONT for

composting, mulching or any other non-agricultural purpose. Do not use animal

waste (e.g. manure, slurry) from animals fed on grass treated with FOREFRONT for

composting or mulching susceptible crops. Kills clover. #

From FOREFRONT label only: Aminopyralid half-life in soils is

dependent on soil type and conditions and is approximately 8-35

days. Rapidly degraded in water

(average half-life 0.6 days).


Active

Ingredient Common Name

Dow Product


Product Persistence and

Degradation (Based largely on data for active ingredient)

Clopyralid Dow Shield* Winter and spring wheat,

barley, oats, linseed, sugar beet, red beet, fodder beet,

mangels, kale, calabrese, broccoli, cauliflower, Brussels

sprouts, fodder rape, swede, turnip, bulb onion, salad onion,

forage maize, sweetcorn, winter

and spring oilseed rape, strawberry, permanent

grassland, rotational grass, ornamental plant production

(directed spray on trees and

shrubs only - avoiding green parts).

Residues in plant tissues (including

manure) which have not completely decayed may affect succeeding susceptible

crops. If treated crop remains have not fully decayed by the time of planting

following crops then avoid planting: peas, beans and other legumes; carrots and

other umbellifers; potatoes; lettuce and

other compositae; glasshouse and protected crops. Straw from treated crops

should not be used or supplied for use as straw in compost, poultry litter, manure or

spent mushroom compost for any

glasshouse crop. Do not apply within the root zone of species of the family

Compositae (e.g. Senecio spp.) or Papillionaceae (e.g. Genista, Cytisus spp.)

The principle route of degradation is

microbial and will occur readily. Half-life in soils is dependent on soil type

and conditions and is approximately 12-62 days.

Picloram Tordon* 22K Land not intended for cropping Sensitive plants may be harmed by

residues of TORDON 22K in soil and treated vegetation. Do not apply TORDON

22K on or adjacent to soil which may be used as garden topsoil, potting soil etc. or

to grass which may be cut and used as a

mulch or for compost for horticultural or garden crops. Do not use cuttings from

treated grass for mulching or composting. TORDON 22K has a negligible effect on

grasses but clover species are highly susceptible.

The major route of degradation is by

photolysis and photodegradation. Microbial degradation leads to

formation of the parent acid which undergoes further degradation. Half

life of the parent acid in soil is

dependent on conditions and is approximately 10-278 days.


Active

Ingredient Common Name

Dow Product


Product Persistence and

Degradation (Based largely on data for active ingredient)

Triclopyr Garlon* 4 Forests and land not intended

to bear vegetation. As a direct treatment on land not intended

to bear vegetation and in forests

None specified Rapidly hydrolysed in soils and water

(with a half-life of less than 1 day) to the parent acid which undergoes

further degradation. Half-life of the parent acid in soil and in water is

dependent on conditions and is approximately 6-52 days.

* Trade Marks # Similar statements appear on the PHARAOH label


Appendix 2 Key UK food, fodder, oil and ornamental crops by plant family and their sensitivity to aminopyralid and clopyralid according to published and unpublished information

Plant Family Food/fodder/oil crops

Ornamental crop examples

Crop labelling indications

Alliaceae Onion Leek

Ornamental alliums

No specified sensitivity to aminopyralid and on-label for clopyralid

Apiaceae (Umbelliferae)

Carrot Celery Parsnip

Angelica Sensitive to aminopyralid and clopyralid

Asteraceae (Compositae)

Lettuce Sunflower

Chrysanthemum Calendula Dahlia Senecio Tagetes (marigold) Zinnia

Sensitive to aminopyralid and clopyralid

Brassicaceae

Cabbage etc. Chinese cabbage Swede Turnip Oil seed rape

Alyssum Aubretia Stock Wallflower

No specified sensitivity to aminopyralid and on-label for clopyralid

Chenopodiaceae Beets Spinach

Mainly weeds

Herbicide carry-over concerns for succeeding crops with aminopyralid (in combination with fluroxypyr) but on-label for clopyralid

Cucurbitae Cucumber Marrow

Gourds No information but generally known as very herbicide-sensitive glasshouse species

Fabaceae (Leguminosae)

Field bean etc. Clover Lentil Pea Soybean

Broom Lupin Mimosa Wisteria


Grossulariaceae Blackcurrant Gooseberry

Flowering blackcurrant Saxifrage

Nothing specific but most likely to be mulched only

Poaceae (Graminae)

Grass Cereals Maize

Ornamental grasses Bamboo

On-label for aminopyralid and clopyralid turf products

Rosaceae

Apple Cherry Pear Raspberry Strawberry

Mainly shrubs and trees Cotoneaster Rose Rowan Spirea

No specified sensitivity to aminopyralid and strawberry is on-label for clopyralid. Also most likely to be mulched


Plant Family Food/fodder/oil crops

Ornamental crop examples

Crop labelling indications

Solanaceae Pepper Potato Tomato

Nicotiana Ornamental pepper Petunia


Notes: 1. The three principal plant families of known high sensitivity to both aminopyralid and

clopyralid and shown in darker blue. The three families of plants tolerant to both these herbicides are uncoloured. The remaining families where there is less certainty or a lack of data are coloured pale blue.

2. These divisions are indicative only and should not be taken as an endorsement by the author to use CGW with known herbicide contamination in any particular situation.

3. There are plants within the other plant families (both listed and unlisted) that are sensitive or tolerant to one or both of these herbicides.

4. Candidate bioassay species underlined


Appendix 3 Estimating potential clopyralid concentrations in UK green compost A theoretical exercise was conducted by the research team with inputs from the Scotts Company UK Ltd. (one of the manufacturers of retail lawn products based on clopyralid) and AfOR to estimate potential levels of clopyralid contamination of compost via treated domestic lawn clippings, based on a variety of expert assumptions. The aim was to estimate the possible range of concentration that might be found in the finished compost products to inform the choice of clopyralid rates to investigate – although the absolute lowest possible concentration would always be zero if none of the clippings had been treated. Factors considered were: 1. Clopyralid application rate 2. Proportion of clippings that had been treated 3. Proportion of herbicide collected and retained by the grass leaves 4. Weight of clippings per cut 5. Number of cuts required to remove all herbicide 6. Moisture loss pre-composting 7. Proportion of grass clippings in compost inputs 8. Mass reduction during composting; and 9. Proportion of herbicide remaining un-degraded at the end of the composting process Three indicative scenarios were considered, the results of which are given in the Table below: 1. The ‘absolute worst case’ is based on the highest recommended rate of application,

100% of clippings treated, 100% capture of the applied herbicide, one cut of just 30g/m2 to remove all herbicide, 50% grass in any one composting batch and no herbicide degradation during composting. These are the most extreme circumstances that can be envisaged.

2. The ‘realistic worst case’ is based on somewhat more realistic assumptions for the maximum: the average recommended rate of application, 35% capture of the applied herbicide, four cuts of 50g/m2 to remove all herbicide, only 7% of clippings treated, 50% grass in any one composting batch and 75% herbicide degradation during composting.

3. The ‘low estimate’ is based on the lowest recommended rate of application, 10% capture of the applied herbicide, ten cuts of 80g/m2 to remove all herbicide, only 1% of clippings treated, 10% grass in any one composting batch and 90% herbicide degradation during composting.

For this exercise it was assumed that: 1. The first four cuts do enter the municipal compost stream (contrary to the latest CRD

labelling requirements not to dispose of any treated grass clippings via council green waste collections) 25 as this is quite likely to happen

2. The herbicide ‘captured’ in the clippings was uniformly divided amongst each cut; so if four cuts was chosen each cut had 25% of the total herbicide in the clippings, treated clippings are uniformly distributed amongst all other clippings and

3. Any herbicide carried on these clippings is uniformly distributed amongst the input materials and of course in the output - although hotspots cannot entirely be ruled out.

25 See http://www.pesticides.gov.uk/approvals.asp?id=2977

http://www.pesticides.gov.uk/approvals.asp?id=2977


The results in Table A1 below suggest that if there was to be contamination, the concentration of clopyralid in CGW on a dry weight basis might range from <1ppb to >1000ppm – a million-fold difference. However, the ‘realistic worst case’ suggests that the concentration encountered would probably not exceed 860ppb – a figure that is towards the upper end of concentrations encountered in the USA as reported by Gilbert et al (see Section 4.1 B of that report).

Table A1. Theoretical estimates of possible clopyralid concentrations in CGW

Units

Clopyralid concentration scenarios

Comments Absolute worst case

Realistic worst case

Low estimate

Herbicide rate applied to domestic lawns (via retail lawn herbicides)

mg ai/m2 10.5 9.0 8.0 Range of recommended rates

Proportion of grass input treated with herbicide

100% 7% 1%

Gilbert et al (2009) data implies perhaps 7% of lawns treated but 1% or fewer is more likely

Herbicide transferred to clippings (total)*

100% 35% 10% Working assumptions

Weight of grass clippings (fresh weight)

g/m2/cut 30 50 80

Scotts information - the greater the weight of clippings the greater the dilution of clopyralid

Number of cuts to remove all herbicide

1 4 10

Ignores CRD label requirement that the first four cuts should not be composted

Proportion of grass mass to compost facility (after moisture loss)

80% 80% 80% Scotts information

Mean herbicide concentration in clippings pre-composting (fresh wt.)

mg ai/kg (=ppm)

438 1.38 0.013 Calculations

Proportion of grass in compost inputs by weight

50% 50% 10% AfOR


Units

Clopyralid concentration scenarios

Comments Absolute worst case

Realistic worst case

Low estimate

Mean herbicide concentration in all compost inputs (fresh weight)

mg ai/kg (=ppm)

219 0.69 0.0013 Calculations

Residual mass after composting

50% 50% 50% AfOR

Residual herbicide as proportion of input level

100% 25% 10.0% Working assumptions

Herbicide concn. in CGW output (fresh weight)

mg ai/kg (=ppm)

438 0.34

Calculations ppb 345 0.25

ppt 250

Herbicide concn. in CGW output (dry weight basis if at 40% MC)

ppm 1094 0.58

Calculations ppb 861 0.625

ppt 625

Herbicide concentration in CGW (if BD is 0.6kg/l)

mg ai/l 263 0.21 0.00015 Calculations

*For grassland it is accepted that a maximum 70% of applied pesticide is captured by foliage. It is assumed that up to 50% of this is retained in leaf tissue. The product of these numbers is 35% and this proportion is taken for the 'likely maximum' It should also be noted that these estimates relate only to clopyralid. It is entirely possible that other synthetic auxin herbicides (such as those being screened at STC) and/or their hormonally active breakdown products may be present at some unspecified concentration(s) in CGW too, and that their effects could be additive.


Appendix 4 Host plant responses to seven concentrations of six herbicides in a peat-based growing medium after 28 days

Host plant Herbicide Herbicide concentration in applied 10ml aliquot

1000 100 10 1 100 10 1 100 10 1 0 ppm

ppm ppm ppm ppm ppb ppb ppb ppt ppt ppt (Control)

Tomato Aminopyralid 3 3 3 2.7 2 0 0 0 0.3 0.3 0

Clopyralid 3 3 2.7 1 0 0 0 0 0.3 0 0

Picloram 3 3 3 2.3 1 0 0.3 0 0.3 0 0

Dicamba 3 3 2.3 1 0 0 0 0 0.3 0 0

MCPA 2.7 1.7 0 0 0 0 0 0 0.3 0 0

2,4-D 2.7 2.3 0.6 0 0 0 0 0 0 0 0

Dahlia Aminopyralid 3 3 3 2 1 0 0.7 0 0.3 0 0.3

Clopyralid 3 3 3 2 0.7 0 0 0 0 0 0

Picloram 3 3 3 2.7 1 0 0.3 0 0.3 0 0

Dicamba 3 3 1 0 1 0 0.3 0 0 0 0

MCPA 2.7 0.7 0 0 0 0 0 0 0 0 0

2,4-D 2.3 0 0 0 0 0 0 0 0 0 0

Lentil Aminopyralid 3 3 3 2 3 0.7 0 0 0 0.3 0

Clopyralid 3 3 3 3 1 1 0 0 0 0 0.7

Picloram 3 3 3 3 2.7 1 0 0.7 0.7 0 0

Dicamba 3 2.3 2.7 0.3 1 0 0 0 0 0.3 0

MCPA 3 3 0 0 0 0 0 0 0 0 0

2,4-D 3 1 0 0 0 1 0 0 0 0 0

Pea Aminopyralid 3 3 3 3 0.7 0 0 0 0.3 0 0

Clopyralid 3 3 3 2.3 0 0 0 0 0 0 0

Picloram 3 3 3 2 0.7 0.3 0 0 0.3 0 0

Dicamba 3 3 1 0 0 0 0 0.3 0 0 0

MCPA 3 3 0.3 0 0 0 0 0 0 0 0

2,4-D 2 0.3 0 0 0 0 0 0 0 0 0

Field bean Aminopyralid 3 3 3 3 2 0.7 1 0.7 0.3 0.7 0.3

Clopyralid 3 3 3 2 0 0 0 0 1 0 0

Picloram 3 3 3 2.3 1 0 0 0 0 0.7 0

Dicamba 3 3 2 0 0 0 0 0 0 0 0

MCPA 3 0 0 0 0 0 0 0 0 0 0

2,4-D 1.3 0 0 0 0.3 0 0 0 0 0 0

Chinese

cabbage Aminopyralid 3 2 1.7 0.3 0.3 0 0 0 0 0 0

Clopyralid 2 0.7 0.3 0 0 0 0 0 0 0 0

Picloram 2.7 2 1 0 0 0 0 0 0 0 0

Dicamba 3 2 1.3 0 0 0 0 0 0 0 0

MCPA 3 2.3 0 0 0 0 0 0 0 0 0

2,4-D 2.7 2 0.3 0 0 0 0 0 0 0 0

Crop response assessed on 0-3 scale

0 = healthy

1 = slight symptoms

2 = moderate symptoms 3 = severe symptoms,

plant dying or dead


Appendix 5 Host plant responses to concentrations of clopyralid from 0-10ppm in a range of Levington M2:CGW mixes after 28 days

Host plant %CGW Herbicide concentration in applied 10ml aliquot

in mix 1000 100 10 1 100 10 1 100 10 1 0 ppm


Tomato 0 - - 3 2 - 0 0 - - - 0.2

12.5 - - 3 2 - 0 0 - - - 0

25 - - 3 2 - 0 0 - - - 0

37.5 - - 3 2 - 0.5 0 - - - 0

50 - - 3 1.8 - 0 0.2 - - - 0

Dahlia 0 - - 3 2 - 0.5 0 - - - 0

12.5 - - 3 2 - 0.2 0.2 - - - 0

25 - - 3 2 - 0.2 0.2 - - - 0.5

37.5 - - 2.8 2.2 - 0.2 0.2 - - - 0

50 - - 2.83 2 - 0.17 0 - - - 0

Lentil 0 - - 3 3 - 1.17 0.67 - - - 0.67

12.5 - - 3 3 - 1.3 1 - - - 0.83

25 - - 3 2.83 - 0.17 0.67 - - - 0.3

37.5 - - 3 3 - 1.5 0.3 - - - 0

50 - - 3 3 - 0.2 0 - - - 0

Pea 0 - - 3 2.3 - 0 0 - - - 0

12.5 - - 3 2.67 - 0 0.5 - - - 0

25 - - 3 3 - 0 0.17 - - - 0

37.5 - - 3 2.3 - 0 0 - - - 0

50 - - 3 3 - 0.3 0 - - - 0

Field bean 0 - - 3 1.8 - 0.83 0 - - - 0

12.5 - - 3 2.6 - 0.4 0.6 - - - 0

25 - - 3 2 - 0 0 - - - 0.2

37.5 - - 3 2.4 - 0.4 0.4 - - - 0

50 - - 3 2.5 - 0 0 - - - 0

Chinese

cabbage 0 - - 0 0 - 0 0 - - - 0

12.5 - - 0 0 - 0 0 - - - 0

25 - - 0 0 - 0 0 - - - 0

37.5 - - 0 0 - 0 0 - - - 0

50 - - 0 0 - 0 0 - - - 0


0 = healthy

1 = slight symptoms

2 = moderate

symptoms

3 = severe symptoms,

plant dying or dead


Appendix 6 Host plant responses to concentrations of six herbicides from 0-1000ppm in aqueous solutions after 7 days Host

plant* Herbicide

Herbicide concentration in aqueous solution

1000 100 10 1 100 10 1 100 10 1 0 ppm


Tomato Aminopyralid 3 3 3 3 2 1 0 0 0 0 0

Clopyralid 3 3 2.3 2 1 0.3 0 0 0.7? 0 0

Picloram 3 3 2.3 2 2 1.7 0 0.3 0 0.3 0.3

Dicamba 3 3 3 2.3 2 0 0 0 0 0 0

MCPA 3 3 2 2 0 0 0 0 0 0 0

2,4-D 3 3 2 1.3 0 0 0 0 0 0 0

Dahlia Aminopyralid 3 1.3 2.3 2.3 2 2 0 0 0 0 0

Clopyralid 3 3 3 2 2 0.3 0 0.7 0 0 0

Picloram 3 2.7 2 2 2 2 0.3 0 0.7 0 0

Dicamba 3 3 2.3 2 0.3 0 0 0 0 0 0

MCPA 2.3 1.7 0.7 0 0.3 0 0 0 0 0 0

2,4-D 3 2.7 2 0.3 0 0 0 0 0 0 0

Lentil Aminopyralid 3 2.7 2.3 2.3 2 2 1 0 0.3 1 0.7

Clopyralid 3 3 2.7 2.7 2.3 1.7 0.3 0.7 0.7 0 0

Picloram 3 3 2.7 2.7 2.7 2 1 1 1 0 0

Dicamba 3 3 2.3 2 2 1 1 0 0 0 0

MCPA 3 2.7 1 0 0 0 0 0 0 0 0

2,4-D 3 3 0.7 0.7 0.3 0.3 0.3 0 0 0 0

Pea Aminopyralid 2.5 3 2 2 1 0 0 0 0 0 0

Clopyralid 3 2.3 2 1.7 0 0 0 0 0 0 0

Picloram 2 2.7 2.7 2.5 1.3 0.7 0.7 0 0 0 0

Dicamba 3 2.3 2 1 0.3 0.3 0 0 0 0 0

MCPA 3 2 1 0 0 0 0 0 0 0 0

2,4-D 3 2.3 2.3 0 0 0 0 0 0 0 0

Field bean Aminopyralid - - - - - - - - - - -

Clopyralid - - - - - - - - - - -

Picloram - - - - - - - - - - -

Dicamba - - - - - - - - - - -

MCPA - - - - - - - - - - -

2,4-D - - - - - - - - - - -

Chinese

cabbage Aminopyralid 2.7 2 1.3 0 0 0 0 0 0 0 0

Clopyralid 1 0 0 0 0 0 0 0 0 0 0

Picloram 3 2.3 1.7 0.7 0 0 0 0 0 0 0

Dicamba 3 2.7 1.7 1 0 0 0 0 0 0 0

MCPA 3 2.3 2 1.3 0.3 0 0 0 0 0 0

2,4-D 3 3 2.3 1.7 0 0 0 0 0 0 0


0 = healthy

1 = slight symptoms

2 = moderate symptoms 3 = severe symptoms,

plant dying or dead

*Field bean was too large for the vials


Appendix 7 Shoot tip enlargement or ‘swelling’ in tomato due to damage from synthetic auxin herbicide

Twisting & distortion of the young leaves of tomato due to damage by synthetic auxin herbicide


‘Pronounced vein’ symptoms in tomato due to damage by synthetic auxin herbicide

Leaf distortion or ‘nettle-like’ symptoms on tomato leaves due to damage by synthetic auxin herbicide


Appendix 8 Leaf-rolling symptoms in field bean due to damage by synthetic auxin herbicide

Severe shoot tip distortion in field bean due to damage by synthetic auxin herbicide


Appendix 9 Shoot tip distortion in dahlia due to damage by synthetic auxin herbicide

Moderate shoot damage symptoms due to damage by synthetic auxin herbicide in dahlia


Appendix 10 Validation trial: Herbicide damage scoring scale for tomato and illustrations of damage 14, 21 and 29 days after sowing Tomato damage scale 0 = no damage with all plants healthy and normal 1 = slight distortion of upper leaves on 1-2 plants per tray 2 = slight/moderate distortion of upper leaves on 3-5 plants per tray 3 = moderate distortion of upper leaves on 6-8 plants per tray 4 = severe stunting with plants not developing beyond the first pair of cotyledon leaves and with severe distortion of any leaves growing from the growing point 5 = all dead


Appendix 11 Validation trial: Herbicide damage scoring scale for field bean and illustrations of damage 14, 21 and 29 days after sowing Field bean damage scale 0 = no damage with all plants healthy and normal 1 = slight distortion of leaves on the stem or some leaf curling 2 = slight/moderate distortion of leaves on the stem or curling on most leaves 3 = moderate distortion of all leaves and reduction in plant height 4 = very severe stunting with plants not developing beyond the first pair of leaves 5 = all dead


Appendix 12 Validation trial: Herbicide damage scoring scale for sunflower and illustrations of damage 14, 21 and 29 days after sowing Sunflower scale 0 = no damage with all plants healthy and normal 1 = slight distortion of leaves on 1-2 plants per tray 2 = slight/moderate distortion of upper leaves on 3-5 plants per tray 3 = moderate distortion of upper leaves and stunting on 6-8 plants per tray 4 = severe stunting with plants not developing beyond the first pair of cotyledon leaves 5 = all dead


Appendix 13 Validation trial: Comparative herbicide damage in tomato 29 days after sowing in media containing composted green wastes of three different EC levels treated with the two highest rates of clopyralid

Clopyralid at 0.0333 and 0.333mg ai/l CGW


Appendix 14 Validation trial: Comparative herbicide damage in field bean 29 days after sowing in media containing composted green wastes of three different EC levels treated with 0 – 0.333mg ai/l clopyralid

Slight to moderate damage on untreated peat + CGW controls

Clopyralid at 0.0000333mg ai/l CGW

Clopyralid at 0.000333mg ai/l CGW Clopyralid at 0.00333mg ai/l CGW

Clopyralid at 0.0333mg ai/l CGW Clopyralid at 0.333mg ai/l CGW


Appendix 15 Validation trial: Comparative herbicide damage in sunflower 29 days after sowing in media containing composted green wastes of three different EC levels treated with the three highest rates of clopyralid




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