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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

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Reference
Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 january 2016 (signature of the study plan) to 30 september 2016 (final report)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
Version / remarks:
April, 2004
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
List of deviations:
1. During breeding worms were fed with oat meal instead of horse manure.
Wrong information was provided in the study plan. This has no impact on the study.
2. Range-finding and the final test: The pH of the acclimatisation and test medium was higher than 6.5, with a maximum of 7.5.
Evaluation: Validity criteria were met, and all worms were in good condition, hence it was assumed not to have affected the results.
3. Range-finding test: there was not enough medium for the determination of soil pH at the end of the test.
Evaluation: pH at the start of the test was determined. Effect parameters were determined in the final test in which pH was determined.
4. Final test: Food was provided on day 20 instead of 21 of the exposure.
Evaluation: The feeding scheme was adjusted because of national holyday.
The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
TEST SUBSTRATE
The artificial soil was prepared according to the guidelines with the following constituents (percentage distribution on dry weight basis) :
- 10% sphagnum peat (Horticoop, Katwijk, The Netherlands);
- 20% kaolin clay (China Clay Ast, source Silex Pottenbakkersmaterialen, ‘s-Hertogenbosch, The Netherlands);
- 70% quartz sand M62 (Eurogrit, Nieuwegein, The Netherlands);
- 0.3-1.0% calcium carbonate (analytical grade, Merck KGaA, Darmstadt, Germany) to obtain an initial pH of 6.0 ± 0.5;
- Milli-RO water to obtain an initial final water content of 40-60% MWHC

Preparation of the test media
The batch of Sodium diisopropylnaphthalenesulphonate tested was a brown fine powder with a purity of 95.1% and sufficiently soluble in Milli-RO water to enable addition to test medium by means of a pre-dissolved stock solution.
A stock solutions was prepared at a concentrations of 5.0 g/L (4.8 g a.i./L, range-finding test) and 5.26 g/L (5.0 mg a.i./L, final test). No other treatment than vigorous shaking was needed to completely dissolve the test substance in Milli-RO water. Lower stock solutions were prepared by diluting the stock in Milli-RO water.
Test media were prepared as indicated in Table 1 and Table 2. All components were mixed (per group) with laboratory mixer for 10-17 minutes. Thereafter, medium was distributed between test vessels. A small amount of medium from each group was left for determination of moisture content and pH. The appearance of media was similar to the control treatment.






Test organisms (species):
Eisenia fetida
Animal group:
other: Earthworm
Details on test organisms:
TEST ORGANISM
- Common name: Eisenia fetida
- Source: Earthworms were bred at the Charles River Den Bosch facilities.
- Age at test initiation: Range-finding test: At least 8 months old with clitellum; Final test: At least 4.5 months old with clitellum
- Weight at test initiation (mean and range, SD): Range-finding test: an individual wet weight: 390-600 mg; Final test: an individual wet weight: 410-600 mg

ACCLIMATION
- Acclimation period: yes
- Acclimation conditions : Seven days prior to the test, earthworms were acclimatized to the artificial soil. The earthworms were fed with the food used in the test.
Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
8 wk
Test temperature:
20-22°C
pH:
The pH was 7.6 at test start and 6.8-7.0 at termination after 56 days.
Moisture:
The soil moisture content was 55-60% at test start and 61-66% at termination after 56 days.
Details on test conditions:
TEST SYSTEM
- Test container (material, size): plastic boxes (18.3x13.6x6 cm) with a perforated lid.
- Amount of soil or substrate/test vessel: 2132 g (artificial soil)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Artificial soil with the following constituents (% distribution on dry weight basis) :
- 10% sphagnum peat (Horticoop, Katwijk, The Netherlands);
- 20% kaolin clay (China Clay Ast, source Silex Pottenbakkersmaterialen, ‘s-Hertogenbosch, The Netherlands);
- 70% quartz sand M62 (Eurogrit, Nieuwegein, The Netherlands);
- 0.3-1.0% calcium carbonate (analytical grade, Merck KGaA, Darmstadt, Germany) to obtain an initial pH of 6.0 ± 0.5;
- Milli-RO water to obtain an initial final water content of 40-60% MWHC

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light
- Light intensity: 426-737 Lux

EFFECT PARAMETERS MEASURED :
- Mortality and adverse effects: day 28
- Body weight: day 0 and 28
- Reproduction: day 56

VEHICLE CONTROL PERFORMED: no

FEEDING
With oat meal moistened with Milli-RO water on day 1, 7, 14, 20 and 28. The food was spread on the surface of the test medium, except on day 28, when food was mixed carefully with the test medium (after removal of the adults) in order to nourish the young worms up to the end of the test. No further feeding took place in the remaining 4 weeks. In case the feeding activity was low, the amount of food was adjusted.

TEST CONCENTRATIONS
Range-finding test: A 14-day range-finding test was performed with procedure and conditions similar to the reproduction test except the following:
- Twenty worms per group (10 per replicate, two replicates) were exposed to the control and concentrations of 0.10, 1.0, 10, 100 and 1000 mg/kg dry soil
- Mortality was assessed on day 7 and 14 of exposure
- Weight was recorded on day 0 and 14 of exposure
Since the observed effects were very subtle, it was decided to increase the spacing factor in the final test to 3 in order to assure that the concentration ranges covers both significant and not significant effects.
Reproduction test:
- 2.0, 6.0, 18, 54 and 162 mg a.i./kg dry soil. A spacing factor of 3 was used in this test as a very shallow dose-response was observed in the range-finding test.
- Control (test medium without test item or other additives).

OTHER:
Introduction of earthworms : After preparation of the test media the earthworms were washed with tap water and introduced onto the surface of the medium.
Test design: Adult earthworms were exposed for 4 weeks and removed on day 28. The cocoons produced were allowed to hatch for a further 4 weeks.
Nominal and measured concentrations:
Range-finding test: Nominal concentrations: 0.10, 1.0, 10, 100 and 1000 mg/kg dry soil
Main test: Nominal concentrations: 0, 2.0, 6.0, 18, 54 and 162 mg a.i./kg dry soil
Reference substance (positive control):
yes
Remarks:
Carbendazim tested (SZBA347XV) 99.2%
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 162 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
4 wk
Dose descriptor:
NOEC
Effect conc.:
>= 162 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth
Duration:
8 wk
Dose descriptor:
NOEC
Effect conc.:
6 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
EC50
Effect conc.:
> 162 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
8 wk
Dose descriptor:
EC10
Effect conc.:
7.4 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
-Survival of adults and adverse effects :
No mortality or clinical effects animals was observed in this test. See details in table n°3.

- Growth of adults :
It should be noted that the weight of worms exposed to the test substance was higher than in the control at the end of the test. See details in table n°4.

- Reproduction :
The reduction of reproduction increased with the applied dose and 36% at the highest concentration tested. A statistically significant effect was observed at the nominal concentration of 18 mg a.i./kg d.s. See details in table n°5.
Results with reference substance (positive control):
- Results with reference substance valid: yes
- Relevant effect levels:
Carbendazim did not reduce the reproduction of Eisenia fetida at a nominal concentration of 1.0 mg/kg d.s. after 56 days of exposure (NOEC).
The 56-day EC50 for reproduction was 1.3 mg/kg d.s. with a 95% confidence interval between 1.0 and 1.8 mg/kg d.s. and complied with the requirements of the guideline. The NOEC for mortality was 1.8 mg/kg d.s. and the NOEC for weight change was 1.0 mg/kg d.s.

Table 3: Mean mortality in the final test

Sodium diisopropylnaphthalenesulphonate

Nominal conc. [mg/kg d.s.]

Total Introduced

Survived

Dead

% Mortality

Control

80

80

0

0.0

2.0

40

40

0

0.0

6.0

40

40

0

0.0

18

40

40

0

0.0

54

40

40

0

0.0

162

40

40

0

0.0

 

Table 4: Mean earthworm weight and weight change at day 28 day of exposure in the final test

Sodium diisopropylnaphthalenesulphonate

Nominal conc. [mg/kg d.s.]

Mean

Std. Dev.

n

% Change1

Control

4.964

0.3032

8

 

2.0

5.087

0.1763

4

-2.5

6.0

5.050

0.1122

4

-1.7

18

5.228

0.3770

4

-5.3

54

5.795

0.5838

4

-16.7

162

5.510

0.2557

4

-11.0

1.    negative numbers indicate increase of weight when compared with the control

 

Table 5: Group mean reproduction and the reduction of reproduction at the end of the test

Sodium diisopropylnaphthalenesulphonate

Nominal conc. [mg a.i./kg d.s.]

Mean

Std. Dev.

n

% Reduction

Control

174.6

22.28

8

 

2.0

187.5

26.89

4

-7.4

6.0

161.8

21.00

4

7.4

18

138.5

30.82

4

20.7*

54

129.8

11.24

4

25.7*

162

111.3

26.60

4

36.3*

*=significantly different from the control (alpha=0.05)

Validity criteria fulfilled:
yes
Remarks:
see conclusions
Conclusions:
Under the conditions of the present study Sodium diisopropylnaphthalenesulphonate did not reduce the reproduction of Eisenia fetida at a nominal concentration of 6.0 mg a.i./kg d.s. after 56 days of exposure (NOEC). The 56-day EC50 for reproduction exceeded the highest concentration tested, i.e. was above 162 mg a.i./kg d.s.

The test was assumed to be valid since the following criteria were met:
- Mean adult mortality in the blank control was ≤ 10% (i.e. 0%).
- Each replicate of the control (containing 10 adults) produced ≥ 30 juveniles by the end of the test (i.e.175).
- The coefficient of variance of reproduction in the control did not exceed 30% (i.e.13%).

Executive summary:

The toxic effects of Sodium diisopropylnaphthalenesulphonate on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a period of eight weeks based on the OECD Guidelines for Testing Chemicals, test No. 222 (April 2004).

The batch of Sodium diisopropylnaphthalenesulphonate tested was a brown fine powder with a purity of 95.1% and sufficiently soluble in Milli-RO water to enable addition to test medium by means of a pre-dissolved stock solution.

A 56-day reproduction test was performed based on the results of a 14-day range-finding test. Forty worms per group (ten per replicate, four replicates) were exposed to 2.0, 6.0, 18, 54 and 162 mg active ingredient (a.i.) per kg dry mass of artificial soil (further referred to as d.s. (dry soil)), whereas 80 worms (ten per replicate, eight replicates) were exposed to an untreated control. The surviving worms were removed after 28 days of exposure and the produced cocoons were allowed to hatch for a period of 28 days. The following endpoints were recorded:

·                    Biomass of adult animals: day 0 and 28

·                    Mortality of adult worms: day 28

·                    Reproduction: day 56

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of the present study Sodium diisopropylnaphthalenesulphonate did not reduce the reproduction ofEisenia fetidaat a nominal concentration of 6.0 mg a.i./kg d.s. after 56 days of exposure (NOEC).

The 56-day EC50for reproduction exceeded the highest concentration tested, i.e. was above 162 mg a.i./kg d.s.

Description of key information

Sodium diisopropylnaphthalenesulphonate did not reduce the reproduction of Eisenia fetida at a nominal concentration of 6.0 mg a.i./kg d.s. after 56 days of exposure (NOEC).

The 56-day EC50for reproduction exceeded the highest concentration tested, i.e. was above 162 mg a.i./kg d.s.

The 56-day EC50 and NOEC of Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda were thus determined by analogy (Read across approach) to be >= 162 mg a.i./kg d.s. 6.0 mg a.i./kg d.s, respectively.

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for soil macroorganisms:
6 mg/kg soil dw

Additional information

The toxicity to soil macroorganisms of Reaction product of naphthalene, butanol, sulfonated and neutralized by caustic soda is determined by a read-across approach based on analogue substance Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda.

A GLP-compliant study (M.A. Tobor-Kaplon, 2016), scored as Klimisch 2 and flagged as a key study, is available on the Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda.

The following results were determined:

Toxicity Metric

Reproduction

Mortality

Weight reduction

 

NOEC

EC10

EC50

NOEC

NOEC

Value[mga.i./kgd.s.]

6.0

7.4

>162

≥162

≥162