Paraquat-dichloride

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

April 1984

Qualifier:

according to guideline

Guideline:

other: EEC guidelines C(L1 )4 Toxicity for Earthworms Artificial Soil Test, Revision 6 prepared for Annex V of EEC Directive 79/831 (Ref. DG Xl/128/82).

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

yes

Remarks:

water

Details on preparation and application of test substrate:

- Preparation of test substance: The substance was prepared in water, made up to 50 mL, from a 200g/L stock solution. This solution was applied to 2.5kg (dry weight equivalents) of artificial soil.
- Method of mixing into soil: The weighed artificial soil was placed into a food mixer bowl and the test solution added slowly while mixing. The soil was mixed for two minutes by a heavy duty food mixer to evenly incorporate the test substance into the soil. Deionised water was then added to bring the moisture content of the soil up to 35 % and mixing continued for a further two minutes.
- Controls: Yes, untreated

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworms
- Age at test initiation: clitellate adults
- Weight at test initiation: 0.30 ± 0.005 g;

ACCLIMATION
- Acclimation period: The test organisms acclimatised in untreated artificial soil for 24 hours

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

20 ± 2 °C

pH:

7.5 at the start of the test

Moisture:

33 to 35%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 litre glass beakers covered with cling film containg six small holes to allow ventilation
- Amount of soil or substrate: 750 g (fresh weight)
- No. of organisms per container: 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SOURCE AND PROPERTIES OF SUBSTRATE
The test soil was a mixture of the following ingredients in oven (approximately 105 °C) dry weight proportions
- 70% fine silica sand (Grade 110)
- 20% kaolinite clay (GTY Powder)
- 10% peat (Humull) organic matter 93.6%
- Calcium carbonate was incorporated into the soil at 10 g/kg

OTHER TEST CONDITIONS
- Photoperiod:24 hour light regime
- Light intensity: 750 ± 250 Lux.

EFFECT PARAMETERS MEASURED
Mortality and any abnormal behavioural effects were assessed after 7 and 14 days in tests. Bodyweight was determined at day 0 and day 14.

VEHICLE CONTROL PERFORMED:
yes

TEST CONCENTRATIONS
- Test concentrations: 1000 mg/kg

Nominal and measured concentrations:

- Nominal: 0 and 1000 mg/kg soil

Reference substance (positive control):

yes

Remarks:

32, 56 and 100 mg/ kg soil chloroacetamide (in water)

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 381 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: pure test substance

Basis for effect:

mortality

Remarks on result:

other: recalculated value, expressed as pure substance, see ‘any other information on results incl. tables’ for respective calculation

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

other: test substance cation species

Basis for effect:

mortality

Remarks on result:

other: Original value presented in study

Details on results:

- Mortality at end of exposure period: There was 2.5% mortality (1 death out of 40) in the treated group and 0% control mortality.
- Changes in bodyweight of live adults at end of exposure period: There was an 18% reduction in bodyweight at 1000 mg/kg ( p= 1% ) , after accounting for control bodyweight loss.
- Morphological and behavioural abnormalities: no abnormalities in behaviour or external condition.
- For details on the results, please refer to the 'any other information on results incl. tables' section.

Results with reference substance (positive control):

The 14 day LC50 value (and 95% confidence limits) for 2-chloroacetamide was 49 (42-52) mg/kg

Reported statistics and error estimates:

- The LC50 value and its 95% confidence limits were estimated for chloroacetamide using the standard technique of iteratively reweighted linear regression of the logit of percent mortality upon log10 (dose).
- The data on bodyweight were analysed by a one-way analysis of covariance using final (post-treatment) bodyweight as the dependent variable and initial (pre-treatment) bodyweight as a covariate.
- LC50 estimation was performed using the in-house package LOGITPC Version 1.2.
- Analysis of covariance was performed using Statistical Analysis System (SAS), Version 6.04.

Table: Number of dead and weight of live E. foetida after 14 days exposure

Substance concentration	Replicate	Number of deaths		Weight of 10 E. Foetida (g)
		7 days	14 days	Start (Day 0)	Finish (Day 14)	% change
Control	1	0	0	3.1	2.5	-19
	2	0	0	3.0	2.9	-3
	3	0	0	3.1	2.8	-10
	4	0	0	3.0	3.0	0
1000 mg/kg	1	1	1	3.1	2.3*	-26
	2	0	0	3.0	2.2	-27
	3	0	0	3.0	2.4	-20
	4	0	0	3.0	2.0	-33

*Where mortality occurred, the end weight and the percentage weight change were adjusted to account for the dead earthworms using the following calculation: End weight for 10 worms (g) = weight of remaining worms (g) x No. Exposed ( 10) /No. Surviving

Table: Treatment means of E. foetida bodyweight after 14 days exposure

Treatment	Mean Adjusted Final Bodyweights and (Standard Errors) for Groups of 10 Earthworms
Control	2.80 (0.09)
1000 mg/kg	2.24 (0.09)**

** Significantly different from control at 1 % level

Calculation of key result

The doses of the test substance were expressed in the test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species: (100/72.4) x 1000 mg test substance cation/kg = 1381 mg pure test substance/kg.

Validity criteria fulfilled:

yes

Conclusions:

Based on the results of this study, the 14-Day LC50 value for the earthworms exposed to the test substance was empirically estimated to be > 1000 mg/kg soil dry weight. This value is corresponding to a recalculated value of 1381 mg/kg pure substance. The 14-day NOEC could not be determined, since significant effects of test substance treatment on weight loss could be determined during the limit-test at a concentration of 1000 mg test substance/kg soil, equivalent to 1381 mg pure substance/kg.

Executive summary:

The toxicity to soil macroorganisms was determined in a study according to OECD guideline 207. In this study, earthworms (4 reps of 10 animals; Eisenia fetida) were exposed to a nominal test substance and limit concentration of 1000 mg test substance/kg soil dry weight for 14 days. Positive (32, 56 and 100 mg/kg chloroacetamide) and untreated controls were included. The test substance was added to artificial soil that contained 70% fine silica sand, 20% kaolinite clay, 10% peat (Humull) organic matter 93.6% and Calcium carbonate was (10 g/kg). The artificial soil had a starting pH of 7.5 and a percent moisture content of 33 to 35%. Test concentrations were not analytically verified and thus test concentrations are given as nominal. Mortality and any abnormal behavioural effects were assessed after 7 and 14 days in tests. Bodyweight was determined at day 0 and day 14. There was 2.5% mortality (1 death out of 40) in the treated group and 0% control mortality and there was an 18% reduction in bodyweight at 1000 mg/kg ( p= 1% ) , after accounting for control bodyweight loss. No abnormalities in behaviour or external conditions were observed. Based on these findings, the 14-day LC50 was determined to be > 1000 mg test substance/kg soil dw, which corresponds to a recalculated value of > 1381 mg/kg soil dw for the pure substance.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 Nov 1971 to 03 Oct 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was applied to field plots on a loamy sand soil, at extremely high rates to simulate the effects of more than one hundred normal applications. 90, 198 and 720 kg cation/ha were incorporated to a depth of 150 mm and earthworm populations assessed 1 and 6 years after treatment. Other plots were treated with 15, 33 and 120 kg cation/ha incorporated to a depth of about 25 mm and earthworm populations assessed 1 year after treatment. For each incorporation depth the low, medium and high rates corresponded to 50, 110 and 400% of the soil's Strong Adsorption Capacity (SAC; 120 μg test substance/g soil).

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

Not reported

Test organisms (species):

other: earthworms (field study)

Animal group:

annelids

Study type:

field study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

72 mo

Remarks:

Observations recorded one and six years after application

Test temperature:

Not reported

pH:

Not reported

Moisture:

Not reported

Details on test conditions:

SITE AND TREATMENT
- Geographic location: The trial was situated near Frensham in Hampshire on a lomy sand soil with a Strong Adsorption Capacity (SAC) for the test substance cation of 120 mg/kg soil.
- Date of treatment: Treatment was made on the 1st and 2nd November 1971.
- Procedure: The trial was divided into 3 blocks. Each block consisted of 2 subblocks, abbreviated S and D, where plots were either Shallow cultivated to a depth of 25 mm, or Deep cultivated to a depth of 150 mm after treatment, respectively. Application rates were calculated to produce 3 concentrations in the soil: 1 = 0.5 x SAC; 2 = 1.1 x SAC and 3 = 4.0 x SAC.

EARTHWORM SAMPLING METHOD
Earthworms were sampled using a formalin expellent method. Each area sampled measured 60 x 60 cm. In 1971, 1972, and 1977, 1, 1 and 2 gallons respectively of 0.2% formalin was applied to each sample area from a watering can. Twenty minutes was allowed for the collection, into a bucket, of expelled earthworms. A little water was added to buckets to reduce irritation to earthworms and maintain them in a healthy condition for live identification. After 20 minutes earthworms were transferred to labelled pots. On return to the laboratory earthworms were weighed, identified and counted. On the 6th September 1971 earthworms were sampled from a single 60 x 60 cm area on each sub-block as a preliminary look, to see what species and numbers of earthworms were present on the site. One year after treatment all plots were again sampled by the method described. Two 60 x 60 cm areas were sampled on each plot, one on the 16th November 1972 (A) and one on the 14th November 1972 (B). Six years after treatment on the 3rd October 1977 3 samples were taken, 1, 2 and 3, from each plot on deep cultivated treatments only, by the formalin expellent method described. Observations on the shallow cultivated plots were discontinued in 1974 following cultivation of plots as part of a management programme to eradicate couch grass Agropyron repens.

Nominal and measured concentrations:

- Nominal application rates deep cultivation (150 mm): 0 (control), 90 (D1), 198 (D2) and 720 (D3) kg cation/ha (corresponding to 0, 50 110 and 400% SAC)
- Nominal application rates shallow cultivation (25 mm): 0 (control), 15 (S1), 33 (S2) and 120 (S3) kg cation/ha (corresponding to 0, 50 110 and 400% SAC)
For both cultivations the theoretical concentration in incorporated depth is 0 (control), 60, 132 and 580 mg/kg soil dw.

Reference substance (positive control):

no

Remarks on result:

other: no effect descriptor reported

Details on results:

One year after application total earthworm numbers on treatments D1, S1 and S2 were not significantly different from controls (P > 5%). Earthworm numbers on plots D2, D3 and S3, 12 months after application were significantly lower than on the control plots, (P ≤ 5%). Six years after treatment complete recovery had taken place on the D2 treatment but not the D3 treatment where a reduction of 35% in total earthworm numbers was still evident when compared with controls (P < 1%). Allolobophora caliginosa and A. chlorotica appeared most responsible for this reduction. However, this was not reflected in total biomass of earthworms which was not significantly different from control (P > 5%).

Reported statistics and error estimates:

Data from all test substance treatments sampled 1 year and 6 years after treatment were analysed statistically by an analysis of variance of log transformed counts. These data were analysed as for a randomised block design with 8 treatments (1st year) or 4 treatments (6 years).

DISCUSSION

The reduction in earthworms on S3, D2 and D3 test substance treatments observed after 1 year, may have been the result of 'free' test substance in the soil. Glasshouse plant bioassays showed that after 1 year soil residues on these plots were still phytotoxic. Six years after treatment it was assumed that populations had reached a state of equilibrium within their treated soil, and reflect the situation which may occur after many years of continual test substance use. During the 5 years following the 1 year post-treatment sampling a complete recovery in earthworm populations had taken place on the D2 plots, and a substantial recovery on D3 plots. How much of this recovery was due to a reduction in mortality, and how much was due to immigration is not clear. However, it is unlikely that immigration could be very effective into a soil which was still toxic to earthworms, unless there was a gradual accumulation of residues leading to mortality. Residue data for the large species Lumbricus terrestris shows that there is little if any test substance in total body tissue after 6 years. The low populations after 6 years, which were confined to the test substance D3 treatments, were associated with phytotoxicity of the crop. This suggests that phytotoxicity may not have been responsible for the lower populations of Allolobophora chlorotica and A. caliginosa on the test substance D3 treatments after 6 years. One year after treatment total number and weight of earthworms on the shallow treated plots (averaged over the test substance rates) was significantly higher (P < 1%) than the deep treated plots. This trend was present at all rates, including controls. Therefore the effect was probably, at least partially, due to the initial rotavation of the deep plots.

Validity criteria fulfilled:

not specified

Conclusions:

On a loamy sand with a low SAC of 120 mg test substance/kg soil the test substance had to be applied at more than a hundred times the normal rate before they affected earthworm populations. Thus the repeated use of the test substance, even on sandy soils, will not affect earthworm populations. The SAC of a soil must be exceeded before the test substance's soil residues affect earthworms.

Executive summary:

The long-term toxicity to earthworms was determined in a field study over 6 years. The test substance was applied to field plots on a loamy sand soil, at extremely high rates to simulate the effects of more than one hundred normal applications. 90, 198 and 720 kg/ha were incorporated to a depth of 150 mm and earthworm populations assessed 1 and 6 years after treatment. Other plots were treated with 15, 33 and 120 kg/ha incorporated to a depth of about 25 mm and earthworm populations assessed 1 year after treatment. For each incorporation depth the low, medium and high rates corresponded to 50, 110 and 400% of the soil's Strong Adsorption Capacity (SAC; 120 μg test substance/g soil). Based on the results, the test substance had to be applied at more than a hundred times the normal rate before they affected earthworm populations. Thus the repeated use of the test substance, even on sandy soils, will not affect earthworm populations. The SAC of a soil must be exceeded before the test substance soil residues affect earthworms.

Description of key information

The 14-d LC50 value is > 1381 mg pure substance/kg soil dw (recalculated) in earthworms (Eisenia fetida), OECD 207, Edwards 1983.

Additional information

Table: Overview of available data on the toxicity to soil macroorganisms except arthropods

Species	Guideline / GLP	Endpoint	Effect value	Comment	Reference
Eisenia fetida	OECD TG 207 / GLP	14-d LC50	> 1381 mg/kg soil dw (recalculated)	Limit test	Edwards, 1993
Earthworms (field; loamy sand)	No guideline specified / GLP not specified	No effect value reported.	No relevant effects observed after 1 and 6 years	The test substance had to be applied at more than a hundred times the normal rate before they affected earthworm populations	Edwards, 1980