Reaction Mass of 3,3-diphenylhexamethyltrisiloxane and 3,3,5,5-tetraphenylhexamethyltetrasiloxane

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16th July 2020 to 17th September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC/MS

Details on sampling:

- Concentrations: All concentrations were analysed: 31.25, 62.5, 125, 250, 500 and 1000 mg a.i./kg dry substrate.
Concentrations of KF-56A were determined by solvent extraction with cyclohexane. Samples were analysed by injection onto a gas chromatography mass spectrometry (GC/MS) system, monitoring three characteristic components.
Samples of each KF-56A concentration were taken for chemical analysis on Days 0, 28 and 56. On Day 0 eight x 5 g dry weight equivalent samples were taken from the bulk mix before the test substrate was distributed between the individual test vessels. On Days 28 and 56, four x approximately 5 g dry weight equivalent samples were taken from each test vessel. On each occasion the samples were weighed into 40 mL glass screw-cap tubes. The Day 28 samples were not analysed immediately but four of the Day 0 samples were analysed on the day of collection and four were retained and stored frozen. The Day 28 and 56 samples were stored frozen following collection, until being transferred to the chemistry department and analysed. Two samples from each replicate of the Day 56 samples were analysed and the others retained. No analysis was performed on the retained samples. Frozen samples were stored for a maximum of one day and were analysed on the day they were removed from storage. Calibration standard solutions were prepared on the day of use.
After analysis the recovery of the individual components was corrected for actual moisture content of soil samples taken from the bulk soil (Day 0) and from the individual replicates on Day 56.
As the reduction in recovery at Day 56 ranged between 1.29 – 14.99% of the analysed content at Day 0, analysis of the Day 28 samples was not undertaken.

Vehicle:

no

Details on preparation and application of test substrate:

Test Substrate
The soil substrate (support medium), allocated batch number Lufa 21/002 was a sandy loam purchased from Lufa Speyer in Germany.
The substrate was stored refrigerated in sealed bags prior to use in the test, and the substrate pH was measured within the two weeks prior to the start of the test to ensure it was within the required range of pH 6.0 ± 0.5 (Actual soil pH 5.59).
The maximum water holding capacity (MWHC) of the substrate was 32.12% and the correct moisture content for the study was determined to be 45% MWHC, equivalent to 14.45% water content.
The procedures for determination of the maximum water holding capacity (MWHC) follow the procedures outlined in OECD Guideline 222, taken from Annex 3 of ISO DIS 11268-2 (1).

Test Design
The test was undertaken using test substance concentrations of 31.25, 62.5, 125, 250, 500 and 1000 mg a.i./kg dry substrate. A water control was also included. Four replicate vessels were prepared for the KF-56A treatments and eight for water control. The test substrate was pre-moistened to 22.5% MWHC two days prior to application of the test substance.

Test System
Each replicate consisted of one 1L glass test vessel containing ten earthworms in the equivalent of approximately 570 g dry weight of soil substrate maintained at nominally 45% maximum water holding capacity (MWHC).
The test substance, corrected for the combined purity of the two main constituents (80.1%), was applied directly into the weighed, pre-moistened substrate and the dose expressed as mg a.i./kg dry substrate. Reverse Osmosis (RO) water was added to achieve the final weight required at 45% MWHC. The soil for the water control vessels was mixed with the same ratio RO water.
Each test concentration was prepared in bulk, and thoroughly mixed for a minimum of four minutes during and after the addition of the test substance and a further ca. five minutes during the water addition using a hand held electric mixer to incorporate both into the test substrate. The water control was mixed as two separate aliquots of 50% of the total weight of substrate required.
Treatments were applied in the order of water control and ascending concentrations of KF-56A.
After mixing, eight 5 g soil samples were taken from each KF-56A treatment and the water control for chemical analysis (four samples to analyse and four to retain). A sample was also taken from each treatment and the control for analysis of water content to enable calculation of the test substance concentration in terms of substrate dry weight.
One sample was taken from each treatment for determination of pH. The remaining soil was then weighed (653 g wet weight, equivalent to ca. 570 g dry weight) into the 1L glass test vessels (diameter ca. 14.6 cm, surface area ca. 167 cm2), which were covered with a plastic film lid.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: Stock cultures with a relatively homogenous age structure (they did not differ in age by more than 4 weeks), from suitable culture stocks purchased from Bias Labs Ltd, UK.
- Age at test initiation (mean and range, SD): between 2 and 12 months old with clitellum
- Weight at test initiation (mean and range, SD): between 300 and 600 mg (wet mass)

ACCLIMATION
- Acclimation period: Prior to the start of the test, the earthworms were acclimatised for at least 24 hours in basic soil substrate. Earthworms were held in a suitable container, and maintained under test conditions.
The earthworms were fed during the acclimatisation period.
- Acclimation conditions (same as test or not): Yes
- Health during acclimation (any mortality observed): No mortality

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

18.6-20.8 °C

pH:

pH of untreated bulk soil substrate 5.59.
pH during test 5.38 to 6.47.

Moisture:

Mean Moisture Content (%): 12.99 to 16.2.
Mean % MWHC: 40.45 to 50.45.
MWHC = 32.12% (100% MWHC = 0.3212g H20/g dry substrate)
To achieve 45% MWHC for 100 g dry substrate: = (100 + ((0.45 x 32.12)) = 114.45 g final weight = 14.45% moisture content
45% MWHC = 14.45% moisture content, 1% moisture content = 45/14.45 % MWHC = 3.1142 % MWHC
%MWHC = 3.1142 x % water content

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1L glass test vessel
- Amount of soil or substrate: 570 g dry weight of soil substrate maintained at nominally 45% maximum water holding capacity (MWHC).
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: n/a

SOURCE AND PROPERTIES OF SUBSTRATE (if soil): Lufa 2.3. Lufa 21/002 was a sandy loam purchased from Lufa Speyer in Germany.
- Geographic location: Rechts der Landauer Str., Nr. 826/7, Offenbach, Rheinland-Pfalz, Germany.
- Pesticide use history at the collection site: Sampling year and former 4 years: none.
- Collection procedures: Sampling date: 12/04/2021
- Sampling depth (cm): 0-20 cm
- Soil texture (if natural soil): not reported.
- % sand:
- % silt:
- % clay:
- Soil taxonomic classification: Silty sand according to German DIN. Sandy loam according to USDA.
- Soil classification system: German DIN and USDA.
- Composition (if artificial substrate): n/a
- Organic carbon (%): 0.66 (+/- 0.09)
- Maximum water holding capacity (in % dry weight): 57.08%
- CEC:
- Pretreatment of soil: drying at room temperature until sieveable (12th to 13th April 2021), presieving 10 mm mesh, final sieving 2 mm mesh. Packaged at 21% MWHP at 28°C.
- Storage (condition, duration):
- Stability and homogeneity of test material in the medium: Stable. 8 Samples were taken for chemical analysis on day 0 from the bulk soil mix. The consistent analytical recoveries indicated that the test material was homogenously distributed.

OTHER TEST CONDITIONS
- Photoperiod: 16:8 hour light: dark conditions,
- Light intensity: 445-732 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Effects on survival and bodyweight and effects on reproduction. Adult survival observations were made daily with final results at day 28. Adult bodyweight was measured at the beginning of the test and at day 28. Number of juveniles was counted at day 56.

VEHICLE CONTROL PERFORMED: n/a

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: control, 10, 100 and 1000 mg/kg dw
- Results used to determine the conditions for the definitive study: Physical effects on adult earthworms in the 100 and 1000 mg/kg dw concentrations and decrease in adult bodyweight in the range-finding test. Effects on reproduction at the

Nominal and measured concentrations:

Nominal: Control, 31.25, 62.5, 125, 250, 500 and 1000 mg a.i./kg dry substrate.
Measured: Control, 32.838, 59.195, 117.329, 298.115, 505.537 and 921.494 mg a.i./kg dry substrate, equivalent to 105.08, 94.71, 93.86, 119.25, 101.11 and 92.15% of the applied dose respectively.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: reported result in terms of 0.66% soil organic carbon

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: reported result in terms of 0.66% soil organic carbon

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: reported result in terms of 0.66% soil organic carbon

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

125 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Adult bodyweight

Remarks on result:

other: reported result in terms of 0.66% soil organic carbon

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Adult bodyweight

Remarks on result:

other: reported result in terms of 0.66% soil organic carbon

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: reported result in terms of 0.66% soil organic carbon

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

62.62 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: confidence limits 33.98, 79.88

Remarks:

reported result in terms of 0.66% soil organic carbon

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

136.9 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Confidence limits 118.4, 158.3

Remarks:

reported result in terms of 0.66% soil organic carbon

Details on results:

- Mortality at end of exposure period: None (table 1)
- Total mass of adults at beginning of test: Approximately 2735.7 mg (table 2)
- Changes in body weight of live adults (% of initial weight) at end of exposure period: See Table 2
- No. of offspring produced: See Table 3
- No. of unhatched cocoons: Not reported
- Morphological and Behavioural abnormalities and Other biological observations: See Table 4. After 28 days of exposure, no adult mortality was recorded in the control or at any KF-56A treatment rate. In the 250, 500 and 1000 mg a.i./kg dry substrate groups earthworms were recorded as agitated (thrashing) when touched and those at 500 and 1000 mg a.i./kg dry substrate were excreting yellow coelomic fluid, a known stress response. The levels of agitation increased with increasing test substance concentration.
The agitation recorded in the 250, 500 and 1000 mg a.i./kg dry soil groups was reflected in the 80.5, 132.9 and 192.6% adult weight loss respectively in comparison to the control.
At Day 56 the mean number of juveniles per vessel was 143.8, 142.5, 75.8, 29.0, 5.3 and 0.5 in the 31.25, 62.5, 125, 250, 500 and 1000 mg a.i./kg dry substrate groups respectively in comparison to 155.5 in the water control. This corresponded to a reduction in juveniles of 7.6, 8.4, 51.3, 81.4, 96.6 and 99.7%, in the 31.25, 62.5, 125, 250, 500 and 1000 mg a.i./kg dry substrate groups, respectively, when compared to the water control. Juvenile worms in the125 mg a.i./kg dry substrate group and above were increasingly agitated when touched gently.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: A laboratory test was conducted in which mature E. fetida were exposed to Carbendazim for a period of 28 days, after which the adult worms were removed.
Any cocoons produced were then allowed to hatch and the juveniles given time to mature for a further 28 days. An adverse effect on adult worm weight over the 28-day exposure period was recorded at 5.0 and 10.0 mg a.i./kg dry substrate.
28-day NOEC value for adult E. fetida survival = 5.0 mg a.i./kg dry substrate
28-day value for adult E. fetida survival >10 mg a.i./kg dry substrate
28-day NOEC value for adult E. fetida weight change 2.5 mg a.i./kg dry substrate
28-day EC50 value for adult E. fetida weight change - 2.865 (2.5 - 3.22) mg a.i./kg dry substrate
NOEC value based on reproduction = 1.25 mg a.i./kg dry substrate
EC10 value based on reproduction = 0.3334 (0.09412 - 1.889) mg a.i./kg dry substrate
EC20 value based on reproduction = 1.311 (N/A - 1.63) mg a.i./kg dry substrate
ECSO value based on reproduction = 2.062 (1.691 - 2.3) mg a.i./kg dry substrate
The 95% confidence limits are presented in parentheses.
The validity criteria were met, and therefore these data are considered to be valid.

Reported statistics and error estimates:

- The NOEC and LOEC for the number of juveniles were determined using a Wilcoxon/Bonferroni-Holm Test.
- Variance in the number of juveniles was evaluated using Bartlett Equality of Variance.
- Distribution was evaluated using the Shapiro-Wilk W Normality test.
- The ECx values were determined using Non-Linear Regression.
- Variances were evaluated using Bartlett Equality of Variance and Mod Levene Equality of Variance
- Distribution was evaluated using the Shapiro-Wilk W Normality test and the Anderson-Darling A2 Normality test

Table 1 Mean Treatment Mortality for Adult E. fetida

	Mortality	Mortality	Mortality
Nominal Concentration (mg a.i./kg dry substrate)	Number of E. fetida Exposed	Day 28 Number of Mortalities	Total %
Water control	80	0	0
31.25	40	0	0
62.5	40	0	0
125	40	0	0
250	40	0	0
500	40	0	0
1000	40	0	0

Table 2 Mean Live Weight of Individual E. fetida and Percentage Weight Change from Day 0 to Day 28

	Live weight (mg)	Live weight (mg)
Nominal Concentration (mg a.i./kg dry substrate)	Day 0	Day 28	Mean change in weight (%)	% Effect in comparison to the water control
Water control	381.7	588.3	+54.98	N/A
31.25	393.8	614.1	+55.96	+1.8
62.5	389.6	608.8	+56.40	+2.6
125	376.9	558.0	+48.11	-12.5
250	400.7	443.2	+10.71	-80.5
500	395.0	319.4	-19.14	-134.8
1000	398.0	195.4	-50.92	-192.6

N/A Not Applicable

Table 3 Mean Number of Juvenile Worms at Day 56

Nominal Concentration (mg a.i./kg dry substrate)	Mean number of juveniles	% reduction when compared to the water control
Water control	155.5	N/A
31.25	143.8	7.6
62.5	142.5	8.4
125	75.8	51.3
250	29.0	81.4
500	5.3	96.6
1000	0.5	99.1

Table 4 Adult E. fetida Observations at Day 28

Nominal Concentration (mg a.i./kg dry substrate)	General observations	Replicate	Replicate specific observations
Water control	Adult worms healthy and have gained weight. Cocoons present	All	-
31.25	Adult worms healthy and have gained weight. Cocoons present	All	-
62.5	Adult worms healthy and have gained weight. Cocoons present	All	-
125	Adult worms healthy and have gained weight. Cocoons present	All	-
250	Significantly less weight gain than in the control. Variation in adult worm weight. Smaller worms agitated when touched.	A	3 of 10 weigh <300 mg
		B	4 of 10 weigh <300 mg
		C	1 of 10 weighs <300 mg
		D	2 of 10 weigh <200 mg, 8 of 10 weigh >300 mg
500	Agitated – agitation increased as the worms were gently blotted dry following washing in RO water. Smaller worms (<300 mg) excreting yellow coelomic fluid – a stress response.	A	All weigh < 300 mg, 8 of 10 weigh <200 mg
		B	8 of 10 weigh <300 mg but >200 mg
		C	2 of 10 weigh ≥200 - <300 mg, 2 of 10 weigh < 200 mg
		D	1 of 10 weighs ≥ 200 - <300 mg, 1 of 10 weighs < 200 mg
1000	Appear a darker red than at lower treatment res and in the control. Clitella less well defined. Agitated – agitation increased as the worms were gently blotted dry following washing in RO water. Smaller worms (<300 mg) excreting yellow coelomic fluid.	A	1 of 10 weighs 200 - <300 mg, 8 of 10 weigh < 200 mg
		B	8 of 10 weigh <200 mg
		C	3 of 10 weigh 200 - <300 mg, 6 of 10 weigh < 200 mg
		D	2 of 10 weigh 200 - <300 mg, 7 of 10 weigh < 200 mg

Table 5 Soil Analysis: Mean Recovery at Days 0, 28 and 56

	Mean recovery (% of nominal)	Mean recovery (% of nominal)
Nominal Concentration (mg a.i./kg dry substrate)	Day 0	Day 56	% loss in recovery at Day 56
Water control	<0.4 mg/kg ^a	<0.4 mg/kg ^b	0
31.25	105.08	89.33	14.99
62.5	94.71	82.34	13.06
125	93.86	82.04	12.59
250	119.25	104.65	12.24
500	101.11	86.73	14.22
1000	92.15	90.96	1.29

^a Component 3 only

^b Components 2 and 3

Validity criteria fulfilled:

yes

Conclusions:

No mortality was observed at the highest test concentration of 1000 mg/kg dry weight. A 28-day NOEC value of ≥1000 mg/kg dry weight has been determined for the effects of the test substance on mortality of adult earthworms (Eisenia fetida).
Effects on bodyweight and reproduction were observed:
A 28-day NOEC value of 125 mg/kg dry weight has been determined for the effects of the test substance on adult bodyweight of Eisenia fetida.
A 56-day NOEC value of 62.5 mg/kg dry weight and an EC10 value of 62.62 mg/kg dry weight have been determined for the effects of the test substance on reproductive output of Eisenia fetida.

Description of key information

28-day NOEC: ≥1000 mg/kg dry weight, for the effects of the test substance on mortality of Eisenia fetida, equivalent to 3030 mg/kg dry weight when normalised to 2% organic carbon.

28-day NOEC: 125 mg/kg dry weight, for the effects of the test substance on body weight of Eisenia fetida, equivalent to 379 mg/kg dry weight when normalised to 2% organic carbon.

56-day NOEC and EC₁₀: 62.5 mg/kg dry weight and 62.62 mg/kg dry weight, respectively, for the effects of the test substance on reproduction of Eisenia fetida, equivalent to 189 and 190 mg/kg dry weight, respectively, when normalised to 2% organic carbon.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

415 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

190 mg/kg soil dw

Additional information

A 56-day earthworm reproduction test (Eisenia fetida) in accordance with OECD TG 222 and in compliance with GLP has been conducted with the registered substance in accordance with ECHA final decision TPE-D-2114497761-36-01/F.

No mortality was observed at the highest test concentration of 1000 mg/kg dry weight. A 28-day NOEC value of ≥1000 mg/kg dry weight (nominal concentration) has been determined for the effects of the test substance on mortality of adult earthworms. Effects on bodyweight and reproduction were observed. A 28-day NOEC value of 125 mg/kg dry weight (nominal concentration) has been determined for the effects of the test substance on bodyweight of adult earthworms. A 56-day NOEC value of 62.5 mg/kg dry weight and an EC₁₀ value of 62.62 mg/kg dry weight (nominal concentration) have been determined for the effects of the test substance on reproductive output of Eisenia fetida (Smithers ERS Ltd., 2021).

E(L)C₅₀ values for effects on mortality and reproduction have also been determined: 28-day LC₅₀ >1000 mg/kg dry weight for mortality, and 56-day EC₅₀ 136.9 mg/kg dry weight for reproduction.

The test was conducted in a natural sandy loam soil with an organic carbon content of 0.66%.

When normalised to 2% organic carbon content, the effect concentrations are as follows:

28-day effect concentrations for mortality:

NOEC: 2% / 0.66% * ≥1000 mg/kg dry weight = ≥3030 mg/kg dry weight.

LC50: 2% / 0.66% * >1000 mg/kg dry weight = >3030 mg/kg dry weight.

28-day effect concentration for body weight:

NOEC = 2% / 0.66% * 125 mg/kg dry weight = 379 mg/kg dry weight.

56-day effect concentrations for reproduction:

NOEC = 2% / 0.66 * 62.5 mg/kg dry weight = 189 mg/kg dry weight;

EC₁₀ = 2% / 0.66% * 62.62 mg/kg dry weight = 190 mg/kg dry weight;

EC₅₀ = 2% / 0.66% * 136.9 mg/kg dry weight = 415 mg/kg dry weight.

Test substance concentrations in the test soil were measured at the beginning and the end of the test, following the single application of the test substance to the soil at the six nominal concentrations of 31.25, 62.5, 125, 250, 500 and 1000 mg/kg dry weight. The measured concentrations at the beginning and end of the test were within 20% of the nominal concentrations therefore the test substance was relatively stable in soil.

In addition to the effects reported on bodyweight and reproduction, behavioural and physical effects were observed. Although after 28 days of exposure, there was no mortality observed in any treatment group, the worms in the 250, 500 and 1000 mg a.i./kg dry substrate groups were recorded as agitated (thrashing) when touched and those at 500 and 1000 mg a.i./kg dry substrate were excreting yellow coelomic fluid, a known stress response. The levels of agitation increased with increasing test substance concentration.

At Day 56, although the juvenile worms that were recovered had moved up to the surface of the substrate during the time the test vessels were in the water bath, those recovered at rates of 250 mg a.i./kg dry substrate and above were increasingly agitated when touched gently. At 250 mg a.i./kg dry substrate the smaller worms were also paler at the posterior end than those from the control and lower treatment rate replicates. At 500 mg a.i./kg two of the 21 juveniles recovered were colourless and overall length was in the range of 7 – 40 mm. The two juveniles recovered at 1000 mg a.i./kg dry substrate were showing normal colouration. There was no visual evidence of dehydration.

Prior to the conduct of the definitive dose response test, a range-finding/limit test was conducted at 10, 100 and 1000 mg/kg dry weight. After 28 days of exposure in the range-finding/limit test, no adult mortality was recorded in the control or in the 10, 100 or 1000 mg a.i./kg dry substrate groups. However, the worms in the 100 and 1000 mg a.i./kg dry substrate groups were recorded as agitated (thrashing) when touched and excreting a yellow bodily fluid. The thrashing motion was particularly violent at 1000 mg a.i./kg dry substrate. Effects on adult body weight were also observed at the 1000 mg/kg concentration.

At Day 56 in the range-finding/limit test, a reduction of 87.9% in the number of juveniles at 1000 mg a.i./kg dry substrate in comparison to the water control was reported. Although the juvenile worms in the 1000 mg a.i./kg dry substrate moved up to the surface of the substrate during the time the test vessel was in the water bath, they were very small and pale. When they were transferred to a hard surface they stopped moving and rapidly dried out. The process of desiccation appeared to start at the tail and rapidly progress towards the head. The juvenile worms were dead within about 15 minutes. When observed under a microscope the dead worms resembled a dried husk, as if all moisture had been lost.

The laboratory have reported that, as a highly lipophilic material which is not water soluble, it is considered possible that the test substance forms a film over the outer cuticle of the worm so preventing the secretion of mucus which keeps the body moist. In the adults this could lead to the irritability noted at Day 28 while resulting in the desiccation of the juveniles recorded at Day 56. It also appears possible that whilst the juvenile worms were in the soil substrate the constant contact with a moist environment prevented the desiccation recorded when they were removed from the substrate and in contact with comparatively dry air.

It is therefore unclear whether the test substance elicits a true toxicological response or whether the adverse observations are a result of physical effects. A precautionary approach is consequently taken and the risk assessment for soil is based on the results of this earthworm test as the starting point for PNEC derivation.