Santicizer P1400

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:

24/12/2021 to 29/04/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Substance Name: Santicizer® Platinum 1400
Abbreviated Name: P1400
Active Substance: Benzyl butyl cis-cyclohexane-1,2-dicarboxylate
CAS Numbers: 1200806-67-2 (generic cis/trans together) and 1931129-39-3 (Cis version).
Purity: 99.886%
Batch/Lot Number: 6660
Receipt Date: 08 March 2021
Expiry Date: 10 February 2024
Appearance: Colourless liquid
Storage on Receipt: Room temperature (15 - 25°C), in darkness

Analytical monitoring:

yes

Details on sampling:

In the range finder test pre-weighed groups of 10 mature E. fetida were exposed to Santicizer® Platinum 1400, incorporated into an artificial soil substrate, for a period of 14 days, at concentrations of 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry substrate. A water control was also included and there were two replicate vessels for the water control and at each treatment rate. Survival was assessed at 7 and 14 days after treatment. The cumulative weight of earthworms remaining for each vessel was also determined at Day 14.

In the definitive test mature E. fetida were exposed to Santicizer® Platinum 1400, incorporated into an artificial soil substrate, for a period of 28 days, at concentrations of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate. After 28 days the adult earthworms were removed from the soil, and the juveniles produced allowed to mature for a further 28 days prior to extraction and counting. A water (untreated) control was included and there were eight replicate vessels for the water control and four vessels for each of the Santicizer® Platinum 1400 treatment rates, each containing ten E. fetida.

Vehicle:

no

Details on preparation and application of test substrate:

The artificial soil substrate was prepared in-house, at Smithers ERS, Harrogate, North Yorkshire, by mixing a batch using the constituents and proportions detailed below.

• 10% Sphagnum peat (2 mm sieved)
• 20% Kaolin clay (kaolinite content above 30%)
• 70% Quartz (silica) sand (predominately fine) with more than 50% of the particles between 50 and 200 microns

The dry ingredients were thoroughly mixed and moisture content was determined by drying a pre-weighed sample at approximately 105ºC. The procedures for determination of the maximum water holding capacity (MWHC) follow the procedures outlined in OECD Guideline 222 (Annex 2), taken from Annex 3 of ISO DIS 11268 2 (1). During the preparation process, calcium carbonate was added as necessary to adjust the substrate to pH 6.0 ± 0.5.
Substrate Batch TTS 21/012, pH 6.26, was used for the acute range finder test. Substrate Batch TTS 22/001, pH 6.27, Maximum Water Holding Capacity (MWHC) 71.05%, was used for the definitive test.
The substrates was stored in closed containers prior to use in the test, and the substrate pH was measured within the three weeks prior to the start of the tests to ensure it was within the required range of pH 6.0 ± 0.5.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

E. fetida is the species of earthworm most widely used in laboratory terrestrial ecotoxicology as a representative of the soil fauna and is a recommended test species as stated in OECD Guideline No 207 and OECD Guideline No 222.
Earthworms were selected arbitrarily from 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. The earthworms were sexually mature (i.e. between 2 and 12 months old with clitellum), weighing between 300 and 600 mg (wet mass).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

56 d

Test temperature:

20 ± 2°C

pH:

The extraction solution (1M KCl) was added to 5 mL sub samples of air-dried at a ratio of 5:1 of extraction solution: dry substrate. These were then shaken for approximately 60 minutes and left to settle for between 1 and 3 hours prior to pH determination using a calibrated pH meter.

Moisture:

For analysis of moisture content at Days 0, 28 and 56 the samples were dried at ca. 105°C, re weighed and the moisture content determined

Details on test conditions:

Lighting and Temperature
The test was carried out in a temperature-controlled room set at 20 ± 2°C and a 16:8 hour light:dark cycle, with light intensity of 400 – 800 Lux, measured weekly throughout the test. Temperature was monitored throughout the test with actual, minimum and maximum values recorded daily.

Moisture Content and pH
For analysis of moisture content at Days 0, 28 and 56 the samples were dried at ca. 105°C, re weighed and the moisture content determined. For measurement of pH, the extraction solution (1M KCl) was added to 5 mL sub samples of air-dried at a ratio of 5:1 of extraction solution: dry substrate. These were then shaken for approximately 60 minutes and left to settle for between 1 and 3 hours prior to pH determination using a calibrated pH meter.

Feeding and Moisture Addition
Food in the form of pasteurised horse manure, and RO water (ca. 5 g and 5 mL, respectively), were added on Day 1 of the test and weekly thereafter up to and including Day 28. Uneaten food remaining in the vessel at each feeding interval was removed and replaced with fresh food. Moisture loss due to evaporation was compensated for on a weekly basis. Each vessel was weighed on Day 1 after the 5 g of food and 5 mL of water were added, to set a “start weight” from which to calculate weight loss the following week. Test vessels were weighed on Days 7, 14, 21 and 28 and the moisture loss due to evaporation compensated for to the nearest 1 mL, if necessary, with the 5 g food and 5 mL water addition. Each vessel was then reweighed to set a “start weight” from which to calculate weight loss the following week. After Day 28, the vessels were weighed weekly and RO water was added to compensate for evaporation loss only.

Nominal and measured concentrations:

Range finder test:
The range finder test was undertaken using test substance concentrations of 0.1, 1.0, 10, 100 and 1000 mg a.i./kg dry substrate).

Definitive test:
Based on the results of the range finder test, the definitive test was conducted at 0, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i. /kg dry substrate (nominal concentrations)
Day 28 measured concentrations were 66.95, 67.77, 49.81, 45.15, 56.15, 54.26, 65.05 and 97.92% of nominal concentrations which were 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i. /kg dry substrate, respectively.
Day 56 measured concentrations 19.13, 11.89, 9.06, 11.87, 7.60, 7.15, 6.08 and 9.52% of nominal concentrations which were 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i. /kg dry substrate, respectively.

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

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

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

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:

other: weight change

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:

other: weight change

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: weight change

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

308.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

555.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

145.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

other: EC20

Effect conc.:

382.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 064.697 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 064.697 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 064.697 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

1 064.697 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: weight change

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 064.697 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: weight change

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

97.555 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

195.468 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

54.65 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

other: EC20

Effect conc.:

125.7 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 476.1 mg/kg soil dw

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Treatment of Results

Rounding Statement
Throughout this study, some of the numerical data presented in the result sections may have been rounded for presentation purposes. Therefore, manual recalculation of the data may result in slightly different values to those shown.

Mortality and Sub-Lethal Effects
Percentage mortality of the worms in each vessel was calculated and a treatment mean presented. A no observed effect concentration (NOEC), a lowest observed effect concentration (LOEC) and an LC50 were determined statistically. The percentage weight change, based on mean surviving worm weight at Day 28 was calculated for each vessel and a treatment no observed effect concentration (NOEC) and the EC50 were determined empirically as there was no weight loss in the Santicizer® Platinum 1400 treatment in comparison to the water control.

Reproductive Output
Juvenile worm numbers were determined for each vessel and a treatment mean presented. A treatment no observed effect concentration (NOEC), lowest observed effect concentration (LOEC) and EC50 values were determined statistically.

Findings
As there were no significant adverse effects on adult earthworm survival or body weight following 14 days exposure to the test substance at rates of up to 1000 mg a.i./kg dry substrate the definitive test to determine the effects on reproduction was conducted at rates of 16.3 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate, a geometric increment of 1.8.

In the definitive test the water control met the validity criteria for adult survival (≥90%), the mean number of juveniles produced per replicate (≥30), and the coefficient of variance for the number of juveniles (≤30%).
After 28 days of exposure, there was no adult mortality recorded in the water control. There was 2.5% adult mortality at 16.3 mg a.i/ kg dry substrate and 0% in all other treatment groups. There were no sub-lethal effects.

The mean number of juveniles per vessel was 123.3, 116.5, 122.3, 124.5, 120.3, 112.8, 97.3 and 83.3 in the 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate treatment groups in comparison to 133.9 in the water control. This corresponded to reductions of 7.9, 13.0, 8.7, 7.0, 10.2, 15.8, 27.4 and 37.8% in the numbers of juveniles at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively in comparison to the water control. The coefficient of variation in the water control was 17.49%.

Analysis of the concentration of Santicizer® Platinum 1400 in the substrate was conducted on samples taken on Days 0, 28 and 56. Post application the mean soil concentrations were equivalent to 111.99, 139.75, 123.30, 132.43, 123.85, 102.02, 110.05 and 115.76% of nominal at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively.
On Day 28 the mean soil concentrations were equivalent to 66.95, 67.77, 49.81, 45.15, 56.15, 54.26, 65.05 and 97.92% of nominal at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively. This represents a decline of 40.22, 51.51, 59.60, 65.91, 54.66, 46.81, 40.89 and 15.41% from the Day 0 concentrations at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively.

On Day 56 the mean soil concentrations were equivalent to 19.13, 11.89, 9.06, 11.87, 7.60, 7.15, 6.08 and 9.52% of nominal at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively. This represents a decline of 82.92, 91.49, 92.65, 91.04, 93.85, 92.99, 94.48 and 91.78% from the Day 0 concentrations at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively.
As the concentration of the test substance in the substrate declined over time the geometric mean concentrations were calculated over 0-28, 28-56 and 0-56 days. The study endpoints have been expressed as both nominal and geometric mean concentrations.

Results with reference substance (positive control):

Toxic Reference
Earthworms from the same source culture as those used in the reproductive test were used in a reference toxicity test with Carbendazim, a known toxic substance (performed under in-house GLP Smithers ERS Study Number 3203131 from November 2021 to January 2022). The EC50 value was calculated to be 3.736 mg/kg dry soil. This is within the given toxicity range of 1 to 5 mg/kg dry soil, indicating that the worms were responding as expected in the test system.

Reported statistics and error estimates:

Statistical Analysis

Statistical analysis of the survival, in comparison to the water control, was conducted using CETIS version 1.8.6.8, based on the nominal test concentrations as mg a.i./kg dry substrate

• The NOEC and LOEC for effects on earthworm survival were determined using a Wilcoxon/Bonferroni Adj Test with ANOVA for detection of significant difference.
• A Grubbs Extreme Value Test detected an outlier at 16.3 mg a.i,/kg dry substrate and the data was analyses both with and without the outlier being included
• Variance was evaluated using a Mod Levene Equality of Variance Test (equal variances) and a Levene Equality of Variance Test (unequal variances).
• Normal distribution was confirmed using the Shapiro-Wilk W Normality test.
• The LCx values were determined using Linear Interpolation (ICPIN), (Two-Point Interpolation).
•

Statistical analysis of the reproduction data, based on both nominal and geometric mean concentrations over Days 0-56, was undertaken using CETIS version 1.8.6.8, based on the nominal test concentrations. The following methods were used:

• The NOEC and LOEC for effects on earthworm reproduction were determined using a Dunnett Multiple Comparison Test with ANOVA for detection of significant difference.
• A Grubbs Extreme Value Test confirmed that there were no outliers in the data set.
• Variance was evaluated using a Bartlett Equality of Variance Test (equal variances).
• Normal distribution was confirmed using the Shapiro-Wilk W Normality test.
• The ECx values were determined using Linear Interpolation (ICPIN), (Two-Point Interpolation).

The geometric mean measured concentrations were calculated using the geomean equation in excel.

Environmental Conditions

Lighting and Temperature

The tests were carried out in a temperature-controlled room under 16:8 hour light:dark conditions, with the light intensity (Lux) measured at least weekly throughout the exposure period. The target light intensity was 400 - 800 Lux.

The temperature range specified in the protocol was 20 ± 2ºC. The light intensity and temperature data recorded over the course of the study are presented below.

Test	Time Period	Temperature ºC		Lighting (Lux)
		Min	Max	Min	Max
Acute range finder	30 December 2021 – 13 January 2022	17.5	21.1	480	776
Definitive	01 March 2022 – 26 April 2022	18.5	20.9	572	794

Range finder

Moisture Content and pH

The pH of the untreated bulk soil substrate was recorded as 6.26 within the three weeks prior to the start of the test. Moisture content and pH of each test concentration were determined at the start and at the end of the test.

Mortality and Sub-Lethal Effects

In the range finder test no mortality was recorded in the water control or for applications of Santicizer^®Platinum 1400 at rates of up to 100 mg a.i./kg dry substrate, with 5% mortality at 1000 mg a.i./kg dry substrate. There was a slight increase in worm weight in the water control and at rates of 0.1, 1.0 and 10 mg a.i./kg dry substrate and slight decreases at 100 and 1000 mg a.i./kg dry substrate. The slight effects recorded at 100 and 1000 mg a.i./kg dry substrate were not considered to be biologically significant. As there were no significant adverse effects on adult earthworm survival or body weight following 14 days exposure to the test substance at rates of up to 1000 mg a.i./kg dry substrate the definitive test to determine the effects on reproduction was conducted at rates of 16.3 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate, a geometric increment of 1.8.

Data from the range finder are summarised in Appendix 3 (attachments)

Definitive

Moisture Content and pH

The pH of the untreated bulk soil substrate was recorded as6.27within the three weeks prior to the start of the test. The moisture content, %MWHC and pH of each test concentration were determined at the start and end of the test. The water content of the substrate had increased in both the control and test substance treatments over the course of the study and was equivalent to >60% MWHC on Days 28 and 56. The data are presented below.

Nominal Concentration (mg a.i./kg dry substrate)	Mean Moisture Content (%)			Mean % MWHC			pH
	Day 0	Day 28	Day 56	Day 0	Day 28	Day 56	Day 0	Day 56
Water control	38.89	45.86	47.67	54.72	64.53	67.07	6.28	6.08
16.3	37.47	45.20	46.26	52.72	63.60	65.09	6.26	6.02
29.4	38.89	46.26	45.73	54.72	65.09	64.34	6.25	6.03
52.9	38.89	45.20	48.15	54.72	63.60	67.75	6.26	5.94
95.3	37.93	42.39	47.34	53.37	59.64	66.61	6.21	5.89
171.5	38.41	42.88	45.99	54.04	60.33	64.71	6.20	5.88
308.6	36.05	44.20	46.26	50.72	62.19	65.09	6.22	5.86
555.6	36.99	45.20	47.34	52.04	63.60	66.61	6.25	5.86
1000	38.89	46.26	47.35	54.72	65.09	66.62	6.23	5.95

MWHC Maximum water holding capacity

MWHC = 71.05% (100% MWHC = 0.7105 g H₂0/g dry substrate)

To achieve 55% MWHC for 100 g dry substrate: = (100 + ((0.55 x 71.05)) = 139.08 g final weight = 39.08% moisture content

55% MWHC = 39.08% moisture content, 1% moisture content = 55/39.08% MWHC = 1.407 % MWHC

%MWHC = 1.407 x %water content

Mortality and Sub-Lethal Effects

The 28-day mortality results forE. fetida exposed to Santicizer^®Platinum 1400 are presented in the table below and in Appendix 5 (Attachments).

Nominal Concentration (mg a.i./kg dry substrate)	Mortality
	Number ofE. fetidaExposed	Day 28 Number of Mortalities	Total (%)
Water control	80	0	0
16.3	40	1	2.5
29.4	40	0	0
52.9	40	0	0
95.3	40	0	0
171.5	40	0	0
308.6	40	0	0
555.6	40	0	0
1000	40	0	0

 

As the concentration of the test substance in the substrate declined over time the geometric mean concentrations were calculated over 0-28, 28-56 and 0-56 days (table below). The study endpoints have been expressed as both nominal and geometric mean concentrations.

Nominal concentration (mg a.i./kg dry substrate)	Mean Measured Soil Concentration (mg a.i./kg dry substrate)			Geometric Mean (mg a.i./kg dry substrate)
	Day 0	Day 28	Day 56	Days 0-56	Days 0-28	Days 28-56
16.3	18.25	10.913	3.118	8.532	14.112	5.833
29.4	41.09	19.924	3.494	14.195	28.613	8.344
52.9	65.23	26.351	4.795	20.200	41.459	11.241
95.3	126.20	43.024	8.725	36.184	73.686	19.375
171.5	212.40	96.298	13.033	64.358	143.016	35.427
308.6	314.83	167.459	17.610	97.555	229.611	54.304
555.6	611.46	361.395	33.797	195.468	470.084	110.517
1000	1157.64	979.216	95.193	476.086	1064.697	305.311

After 28 days of exposure, no adult mortality was recorded in the water control with 2.5, 0, 0, 0, 0, 0, 0 and 0% mortality at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively in the Santicizer^®Platinum P1400 treatments. There were no sub-lethal effects.

Mean earthworm weight change data are presented in the table below and Appendix 6 (Attachments)

Nominal Concentration (mg a.i./kg dry substrate)	Mean Live Weight (mg)		Change (%)
	Day 0	Day 28
Water control	487.9	464.3	-4.83
16.3	466.7	515.6	+10.49
29.4	479.7	491.0	+2.36
52.9	472.8	490.4	+3.72
95.3	479.6	491.5	+2.49
171.5	480.9	460.6	-4.24
308.6	477.9	475.6	-0.47
555.6	471.0	475.6	+0.98
1000	486.4	479.6	-1.41

At Day 28, there were mean weight gains of 10.49, 2.36, 3.72, 2.49 and 0.98% at nominal concentrations of 16.3, 29.4, 52.9, 95.3 and 555.6 mg a.i./kg dry substrate respectively with weight reductions of 4.24, 0.47 and 1.41% at 171.5, 308.6 and 1000 mg a.i./kg dry substrate respectively in the Santicizer^®Platinum 1400 treatments in comparison to a reduction of 4.83% in the water control. The effect in comparison to the water control has not been calculated as the slight changes in weight over the 28 day exposure period were not considered to be biologically significant.  There were no other observations noted.

Based on these results, the NOEC and LOEC for 28-day adult survival were determined to be 1000 and >1000 mg a.i./kg dry substrate respectively, and the LC₅₀for 28-day survival was statistically determined to be >1000mg a.i./kg drysubstrate. The NOEC for overall weight change was determined to be 1000 mga.i./kg dry substrate. The EC₅₀value for weight change was empirically determined to be >1000 mga.i./kg dry substrate.

Based on the geometric mean concentrations over Days 0-28, the period for which the adult worms were exposed,the NOEC and LOEC for 28-day adult survival were determined to be 1064.697 and >1064.697 mg a.i./kg dry substrate respectively, and the LC₅₀for 28-day survival was empirically determined to be >1064.697 mg a.i./kg dry substrate. The NOEC for overall weight change was determined to be 1064.697mga.i./kg dry substrate. TheEC₅₀value for weight change was empirically determined to be >1064.697mga.i./kg dry substrate.

Reproduction

The number of juvenile worms in each test vessel was counted and the data are presented in the table below and Appendix 7 (Attachments)

Nominal Concentration (mg a.i./kg dry substrate)	Mean Number of Juveniles	Mean Number of Cocoons	% Difference in Mean Number of Juveniles when Compared to the Water Control^a
Water control	133.9	0.6	N/A
16.3	123.3	0.3	-7.9
29.4	116.5	0.3	-13.0
52.9	122.3	0.3	-8.7
95.3	124.5	0.3	-7.0
171.5	120.3	0	-10.2
308.6	112.8	0.3	-15.8
555.6	97.3	0.3	-27.4
1000	83.3	0	-37.8

The mean number of juveniles per vessel was 123.3, 116.5, 122.3, 124.5, 120.3, 112.8, 97.3 and 83.3 at nominal concentrations of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively in the Santicizer^®Platinum 1400 treatments in comparison to 133.9 in the water control. This corresponded to reductions in juveniles of 7.9, 13.0, 8.7, 7.0, 10.2, 15.8, 27.4 and 37.8% at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively, compared to the water control. There were no other observations noted. Based on these results, the NOEC for reproduction was statistically determined to be 308.6mg a.i./kg dry substrate with a LOEC of 555.6 mg a.i./kg drysubstrate. The EC₁₀, EC₂₀and EC₅₀value for reproduction were statistically determined to be 145.1 (N/A, 449.1), 382.4 (N/A, 785.4) and >1000 mg a.i/kg dry substrate respectively.

The statistics reports are presented in Appendix 8 (Attachments).

Based on the geometric mean concentrations over Days 0-56, the NOEC for reproduction was statistically determined to be 97.555 mg a.i./kg drysubstrate with a LOEC of 195.468 mg a.i./kg dry substrate.The EC₁₀, EC₂₀andEC₅₀values for reproduction were statistically determined to be 54.65 (N/A, 141.9), 125.7 (N/A, 322.3) and >476.1 mg a.i./kg dry substrate respectively.

A summary of all endpoints is presented in the table below.

 

Endpoint	Nominal Concentration (mg a.i./kg dry substrate)	Geometric mean (mg a.i./kg dry substrate)
28-day NOEC Adult Survival	1000^b	1064.697^a
28-day LOEC Adult Survival	>1000^b	>1064.697^a
28-day LC50Adult Survival	>1000^b	>1064.697^a
28-day NOEC Adult Weight Change^a	1000	1064.697
28-day EC50Adult Weight Change^a	>1000	>1064.697
56-day NOEC Reproduction^b	308.6	97.555
56-day LOEC Reproduction^b	555.6	195.468
56-day EC10Reproduction^b	145.1 (N/A, 449.1)	54.65 (N/A, 141.9)
56-day EC20Reproduction^b	382.4 (N/A, 785.4)	125.7 (N/A, 322.3)
56-day EC50Reproduction^b	>1000	>476.1

Chemical analysis

The analytical procedure is presented in Appendix 10 ( Attachments)

Post application the mean soil concentrations were 18.25, 41.09, 65.23, 126.20, 212.40, 314.83, 611.46 and 1157.64 mg a.i./kg dry substrate at application rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6and 1000 mg a.i./kg dry substraterespectively. This is equivalent to 111.99, 139.75, 123.30, 132.43, 123.85, 102.02, 110.05 and 115.76% of nominal at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6and 1000 mg a.i./kg dry substrate respectively.  Santicizer^®Platinum 1400was not detected in the water control samples. Moisture content of the samples was corrected using the mean moisture content of the appropriate treatment rate.

On Day 28 the mean soil concentrations were 10.913, 19.924, 26.351, 43.024, 96.298, 167.459, 361.395 and 979.216 mg a.i./kg dry substrate at application rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively. This is equivalent to 66.95, 67.77, 49.81, 45.15, 56.15, 54.26, 65.05 and 97.92% of nominal at16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000mg a.i./kg dry substrate respectively. This represents a decline of 40.22, 51.51, 59.60, 65.91, 54.66, 46.81, 40.89 and 15.41% from the Day 0 concentrations at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substraterespectively. Santicizer^®Platinum 1400was not detected in the water control samples. The moisture content was adjusted for each individual sample.

On Day 56 the mean soil concentrations were 3.118, 3.494, 4.795, 11.314, 13.033, 22.056, 33.797 and 95.193 mg a.i./kg dry substrate at application rates of16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000mg a.i./kg dry substrate respectively. This is equivalent to 19.13, 11.89, 9.06, 11.87, 7.60, 7.15, 6.08 and 9.52% of nominal at16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry substrate respectively. This represents a decline of 82.92, 91.49, 92.65, 91.04, 93.85, 92.99, 94.48 and 91.78% from the Day 0 concentrations at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6and 1000 mg a.i./kg dry substrate respectively. Santicizer^®Platinum 1400was not detected in the water control samples. The moisture content was adjusted for each individual sample.

A summary of the mean recovery at Days 0, 28 and 56 with the % decline at Days 28 and 56 is presented in the table below and the individual replicate data in Appendix 9 (Attachments).

Nominal concentration (mg a.i./kg dry substrate)	Mean recovery (% of nominal)			% decline from analysed content at Day 0
	Day 0	Day 28	Day 56	Day 28	Day 56
Water control	0	0	0	0	0
16.3	111.99	66.95	19.13	40.22	82.92
29.4	139.75	67.77	11.89	51.51	91.49
52.9	123.30	49.81	9.06	59.60	92.65
95.3	132.43	45.15	11.87	65.91	91.04
171.5	123.85	56.15	7.60	54.66	93.85
308.6	102.02	54.26	7.15	46.81	92.99
555.6	110.05	65.05	6.08	40.89	94.48
1000	115.76	97.92	9.52	15.41	91.78

Validity criteria fulfilled:

yes

Conclusions:

The 28 day LC50 value for adult E. fetida survival is >1064.697 mg a.i./kg dry substrate
The 56 day EC10 value for effects on reproduction is 54.65 (N/A, 141.9)a mg a.i./kg dry substrate

Executive summary:

A laboratory test was conducted in which mature E. fetida were exposed to Santicizer^®Platinum 1400  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. There were no adverse effects on adult worm survival or bodyweight over the 28-day exposure period. The validity criteria were met, and therefore these data are considered to be valid. The concentrations ofSanticizer^®Platinum 1400 in the substrate declined over the 56-day exposure period.

Description of key information

A laboratory test was conducted in which matureE. fetidawere exposed to Santicizer^®Platinum 1400 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.There were no adverse effects on adult worm survival or bodyweight over the 28-day exposure period. The validity criteria were met, and therefore these data are considered to be valid. The concentrations ofSanticizer^®Platinum 1400 in the substrate declined over the 56-day exposure period.

The 28 day LC50 value for adult E. fetida survival is >1064.697 mg a.i./kg dry substrate (mean measured concentration)

The 56 day EC10 value for effects on reproduction is 54.65 (N/A, 141.9)a mg a.i./kg dry substrate (mean measured concentration)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 064.697 mg/kg soil dw

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

54.65 mg/kg soil dw