4,4'-sulfonylbisphenol, polymer with ammonium chloride(NH4Cl), pentachlorophosphorane and phenol

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:

16 Aug 2022 - 11 Oct 2022

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))

Version / remarks:

2016

Deviations:

yes

Remarks:

The test was performed under continuous lighting instead of light-dark cycles of 16h:8h, as prescribed by OECD TG 222. See 'Details on results' for evaluation of this deviation.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Appearance: Pale yellow solid
- Storage condition: Room temperature in the dark

Analytical monitoring:

not required

Vehicle:

yes

Remarks:

Quartz sand

Details on preparation and application of test substrate:

As the test item is not soluble, dispersible or emulsifiable in water, the test item was mixed with fine quartz sand (10 g of a mixture of quartz sand and quantity of test substance corresponding to 600 g dry weight of artificial soil, source quartz sand: Gebrüder Dorfner GmbH Co., Kaolin- und Kristallquarzsandwerke KG, Scharhof 1, D-92242 Hirschau, Germany) and then applied with this mixture to artificial soil and then moistened, using a laboratory mixer, to ensure a homogeneous distribution. The control was treated with the same amount sand as the test item groups. The soil for the treatment concentration and for the control was prepared in two batches, which was then split into the replicates.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: manure worm
- Strain code: VT BASF 05 09 289 – 290 – 291
- Source: originally obtained from BASF SE Crop Protection Division, Limburgerhof, Germany. Bred under standardised conditions in the Ecotoxicological Laboratory of the Test Facility.
- Age at test initiation: About 5 months (not differing in age by more than 4 weeks) with clitellum.
- Weight at test initiation: 300 - 592 mg

ACCLIMATION
- Acclimation period: 1 day
- Acclimation conditions: in artificial soil under test environmental conditions

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

56 d

Remarks:

28 d adult exposure, additional 28 d for reproduction assessment

Test temperature:

19.7 - 22.0 °C

pH:

Control soil: 6.33 - 7.39
Limit concentration soil: 6.39 - 7.42

Moisture:

Water content at test start: 21.5-22.1% of soil dw (approx. 58.8% of maximum WHC)
Water content at test end: 23.9-25.7% (approx. 67% of maximum WHC)

Details on test conditions:

TEST SYSTEM
- Test container: plastic containers with ~1 L volume and ~200 cm2 area, covered with transparent and punctured lid to allow gas exchange
- Amount of soil or substrate: 719 g moist artificial soil, corresponding to 600 g soil dry weight
- No. of organisms per container: 10
- No. of replicates per treatment group: 8
- No. of replicates per control: 8
- The test containers were held in a climate chamber under continuous lighting with at a light intensity of about 442.8 lux.
- On day 0, after washing and drying gently, the individual weight of each worm was determined; then worms were exposed by placing on the surface of the treated artificial soil.
- Feeding: One day after the start of the test, earthworms were fed with approximately 5 g air-dried finely ground cow manure (obtained from cows that were not subject to treatment with veterinary products that could adversely affect the worms during the test) per container. It was spread on the soil surface of the containers and moistened with approximately 5 to 6 mL deionised water. Food was provided once a week during the first 4-week period of the test. After adults were removed from the soil on day 28 of the test, a further 5 g of food was administered to each test container. No further feeding was done during the remaining 4 weeks of the test.

ARTIFICIAL SOIL SUBSTRATE
Artificial soil was prepared, as follows:
- 5 % sphagnum peat (as close to pH 5.5-6.0 as possible), no visible plant remains, finely ground, dried to measured moisture content)
- 20 % kaolin clay (kaolinite content above 30 %)
- 75 % industrial sand (fine sand is dominant with more than 50 % of the particles between 50 and 200 μm)
The air-dry constituents (sphagnum-peat, kaolin clay and sand) were blended in the correct proportions and mixed thoroughly with deionised water. The final water content was equivalent to about 60 % of the maximum WHC. The determination of pH value was done according to ISO 10390.
The pH of the treated artificial soil was determined at the beginning, and at the end of the test.
The maximal water holding capacity (WHC) of the artificial soil was determined according to OECD 222 Guideline.
The moistened soil was divided in test containers corresponding with the number of test concentrations and controls.
Soil moisture content was determined at the beginning and at the end of the test by a SARTORIUS Moisture Analyzer.
The water content of the artificial soil before moistening was 1.92 g / 100 g dry soil. The maximal water holding capacity of the artificial soil was 37.1 g water / 100 g soil dry weight.


EFFECT PARAMETERS MEASURED
- Mortality: On day 28 of the experiment the adult worms were removed from the soil and the number of live adult worms was recorded. The mortality was assessed by emptying the test medium onto a suitable tray, sorting worms from the medium and testing their reactions to touch. Earthworms were considered dead if they fail to respond to gentle stimulation at the front end. Due to rapid decomposition under test conditions, missing earthworms were considered dead. Living adult worms were euthanized by rapid freezing at ~-80°C.
- Clinical symptoms: Any symptoms of earthworms observed without disturbing the soil during the exposure period was recorded.
- Biomass: The live weight of the adult worms was determined after gentle washing and drying in each test container on Day 0 and 28 of the experiment.
- Feed consumption: The feed consumed was estimated visually. When food consumption was low, feeding was reduced.
- Number of juvenile worms: On day 56 of the experiment the juvenile worms hatched from cocoons were separated from the soil (using a water bath at a temperature of about 60°C) and counted, then euthanized by rapid freezing at ~-80°C.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Range finding study: performed with 1 replicate (10 worms) for concentrations 1000, 333, 100, 10 and 1.0 mg/kg soil dry weight plus a control. Mortality and biomass were assessed after 14 days.
- Results used to determine the conditions for the definitive study: no mortality or effects on body weight were observed, therefore the final test was performed as a limit test at 1000 mg/kg soil dry weight.

Nominal and measured concentrations:

Control and 1000 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Carbendazim, tested in a separate study (study code: 21/011-211G)

Key result

Duration:

56 d

Dose descriptor:

NOEC

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.:

>= 1 000 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:

growth

Remarks:

Based on body weight

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

Details on results:

Deviation from OECD guideline 222:
According to the relevant guideline (OECD No.222) the test should be carried out under controlled light-dark cycles (preferably 16 hours light and 8 hours dark). Because of technical reasons (the earthworms try to escape from the test vessels in the dark, with illumination the worms remain in the soil) the test was performed under continuous lighting according to the experience of this laboratory. This deviation is considered to be justified on scientific grounds and to have no impact on the outcome of the study or the interpretation of the results (under the soil, the earthworms remain in dark conditions).

See Table 1 for a summary of the results.
- Mortality: there was no mortality in the control or in the treatment group after 28 days. Therefore, the LC50 value was determined to be higher than 1000 mg/kg soil dry weight and the NOEC was determined as 1000 mg/kg soil dry weight.
- Clinical symptoms: No behavioural effects were observed during the experiment in the control group.
- Biomass: The biomass increase at day 28 was 23.8% in the control group and 24.0% in the treatment group. The changes of biomass development in the treatment group were statistically not significant compared to the control group.
- Food consumption: No test item related effects were detected on food consumption. The average food consumption was 25.0 g in the control and test item treated group.
- Number of juveniles: The mean number of offspring was 124.3 juveniles (SD: 17.2) per replicate in the control and 126.8 (SD: 26.0) in the treatment group. The number of juveniles was statistically not significantly different at the concentration level of 1000 mg/kg soil dry weight compared to the control. Therefore, the EC50 was determined to be higher than 1000 mg/kg soil dry weight and the NOEC was determined as 1000 mg/kg soil dry weight.

Results with reference substance (positive control):

The sensitivity and reliability of the experimental procedure is assessed once a year by use of an item which is known to be toxic and have an inhibitory effect to the earthworm reproduction such as Carbendazim (Active ingredient content: 99.3 %) (OECD 222, adopted in 2016). Study Code of the last study: 21/011-211G, the EC50 after 56 days was determined to be 1.43 mg Carbendazim/kg soil dry weight (95 % confidence limits: 1.34 – 1.53 mg a.i./kg soil dw). The reference value range is 1 – 5 mg a.i./kg dry mass, hence the results in this laboratory are considered to be fully acceptable.

Reported statistics and error estimates:

For adult worms, mean weight values per container, and standard deviations were calculated for each treatment and each replicate at the start and on the 28th day of the test using Excel for Windows software.
For juveniles, mean number of animals and standard deviations were calculated for each treatment on the 56th day of the test using Excel for Windows software.
The EC/LC values of the test item and its confidence limits were calculated using ToxRatPro software. Statistical analysis of feed consumption was assessed by Duncan test (p < 0.01) by SPSS/PC+ statistical program. Statistical analysis of the number of juveniles, mortality and the worm biomass were assessed by analysis of variance (ANOVA), William’s test, Shapiro-Wilk’s test, Levene test ( = 0.05, one sided-smaller) and Probit analysis or logistic regression by ToxRatPro statistical program. The No-Observed-Effect-Concentration (NOEC) and the Lowest-Observed-Effect-Concentration (LOEC) was determined. The premises of ANOVA (homogeneity of variance) was tested by adequate test statistics.

Table 1: Summary of results

Concentration of Test Item [mg/kg soil dry weight]	Mortality at 28 d [%]	Weight change at 28 d Mean (SD) [%]	Number of juveniles per replicate Mean (SD) [#]
Control	0	23.8 (5.9)	124.3 (17.2)
1000	0	24.0 (8.5)	126.8 (26.0)

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

Under the conditions of the test, the test item had no toxic effect on the mortality, growth and reproduction of earthworms (Eisenia fetida) at the limit concentration (1000 mg/kg soil dry weight). Overall NOEC: 1000 mg/kg soil dry weight.

Executive summary:

In a chronic toxicity study, earthworms (Eisenia fetida) were exposed to SPS-100 for 28 days in artificial soil (according to OECD 222, with 5% peat). After this period, the adults were removed and cocoons remained in the test vessels for additional 28 days (56 days total study duration). The test was conducted in plastic boxes covered with perforated lids, containing 600 g artificial dry soil. Based on a range-finding study, the final study was conducted as a limit test at a nominal concentration of 1000 mg/kg soil dry weight and an untreated control. Both groups had 8 replicates of 10 worms each.

No mortality was not observed in the treatment group or control. Body weight change in the treatment group after 28 days was not statistically different from control. The reproduction rate in the treatment group was not statistically significantly different compared to the control. No abnormal behaviour or changes in food consumption were observed.

The NOEC values for mortality at 28 d, body weight change at 28 d and reproduction at 56 d were 1000 mg/kg soil dry weight. The EC50 value for reproduction and the LC50 value for mortality were >1000 mg/kg soil dry weight. The study met all validity criteria and is considered to be reliable without restriction.

Description of key information

The test item had no toxic effect on the mortality, growth and reproduction of earthworms (Eisenia fetida) at the limit concentration (1000 mg/kg soil dry weight). Overall NOEC: 1000 mg/kg soil dry weight.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw