Reaction products of diphenylamine with nonene, branched

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:

2014

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

Analytical monitoring:

yes

Details on sampling:

Samples were collected on Day 0, at the time of soil preparation, to determine homogeneity, measure test concentrations and establish Day 0 values for evaluating stability over the course of the exposure period. On Day 0 a set of back up samples was collected to provide extra samples in the event that they were needed to complete analysis. On Day 56, samples were collected from one replicate of the negative and solvent control groups and two replicates of the low, middle and highest test concentrations.

The artificial soil samples were collected from the negative and solvent controls and 62.5,
125, 250, 500 and 1000 mg/kg dry soil test concentrations on Day 0 and stored frozen until analysis. Three samples from the top, middle, and low layer of soil were collected for the concentrations of 62.5 and 1000 mg/kg dry soil to evaluate homogeneity. Samples were collected from the negative and solvent control groups as well as the 62.5, 250 and 1000 mg/kg dry soil test concentrations on Day 56 and stored frozen until analysis. Samples were removed from frozen storage and placed at ambient temperature to thaw before extraction.

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

Test soil was prepared by mixing the appropriate amount of test substance first with acetone then with dry artificial soil before adding reverse osmosis water to hydrate the soil. Test soil components were mixed for up to a total of 20 minutes in order to achieve a homogeneous mixture. Negative control soil was prepared in a similar manner as the treated soil but without the addition of test substance or solvent. Solvent control soil was prepared with acetone but without test substance. 750 g of prepared soil were added to each of four test chambers for each of the treatment and solvent control groups, the negative control group contained 8 test chambers.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

- Common name: earthworm- Origin: Wildlife International In-house culture started with earthworms obtained from: University of Maryland, Wye Research & Education Center, Queenstown, MD 21658; Specifics: Only adult worms (with clitellum) were used. - Breeding: Earthworms were from synchronous cultures (individuals not differing in age by more than four weeks) maintained in moist peat moss and fed saturated alfalfa and/or cow manure. - Acclimatization: Adult earthworms were transferred to the test room and held in a glass container for acclimation to test conditions for a week prior to test initiation. One day prior to the test the worms (380 with clitellum) were removed from the chamber and divided into ten one-liter glass beakers containing prepared artificial soil substrate adjusted to a moisture content of approximately 34% by weight, for the soil acclimation period. Earthworms were fed cow manure throughout the acclimation period.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

Start: 19.8 - 20.1, End: 20.5 - 20.7°C

pH:

Start: 7.0 - 7.2, End: 7.2 - 7.3

Moisture:

Start: 33 - 35%, 36 - 39 %

Details on test conditions:

TEST SYSTEM- Test substrate: 70% sand, 20% kaolin clay, 10% sphagnum peat and 0.01% calcium carbonate
Total mass of the test substrate: 8 kg for the control groups, 4 kg for each treatment group (wet weight; soil moisture content approximately 34 %. (mean value).-
Test units: one-liter glass beakers covered with plastic wrap
Test substrate per test container: Approx.750 g artificial soil (wet weight)-
Number of animals / test container: 10-
Worm weight at start of exposure: In a range of 250 - 600 mg-
Number of replicates: 8 replicates for control and 4 replicates for each test substance concentration as well as the solvent control-
Number of test substance concentrations: 5-
Illumination: Light/day cycle of 16:8 hours, over the exposure period-
Light intensity: 504 +-60.5 lux-(range: 401 – 613 lux)
Feeding: The earthworms were fed cow manure during testing. On Day 1 of the test, 5 grams of finely ground cow manure for food, and water to moisten the food, were added to the test chambers. Food was provided approximately weekly during the next three weeks by adding food in a small depression in the soil surface and covering it with a thin layer of soil. The amount of food supplied was reduced if uneaten food remained from the previous feeding interval. On Day 28, after adult earthworms were removed, approximately 5 additional grams of manure was gently mixed into the test soil before it was returned to the test chambers.
Preparation: The artificial soil was prepared in bulk by blending 70% sand, 20% kaolin clay, 10% sphagnum peat and 0.01% calcium carbonate. The pH of the bulk soil at the completion of preparation (prior to hydration) was 5.9. The bulk artificial soil was stored in a sealed container under ambient conditions until used to prepare the test soils. Test soil was prepared by mixing the appropriate amount of test substance first with acetone then with dry artificial soil before adding reverse osmosis water to hydrate the soil. Test soil components were mixed for up to a total of 20 minutes in order to achieve a homogeneous mixture. Negative control soil was prepared in a similar manner as the treated soil but without the addition of test substance or solvent. Solvent control soil was prepared with acetone but without test substance. 750 g of prepared soil were added to each of four test chambers for each of the treatment and solvent control groups, the negative control group contained eight test chambers.
Insertion of earthworms: On the day of test initiation, the acclimatised earthworms were rinsed briefly with well water, blotted on paper, weighed in groups of 10, and then placed on the soil surface of a test chamber.

Nominal and measured concentrations:

nominal: 62.5, 125, 250, 500 and 1000 mg/kg dry soil

Reference substance (positive control):

yes

Remarks:

Carbendazim

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

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

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:

other: body weight

Details on results:

No significant effects were noted at any of the tested concentrations.

There were no mortalities in the control and treatment groups during the 28 day adult exposure period, with the exception of one dead earthworm in the 1000 mg/kg dry soil group. The one dead earthworm was considered to be an incidental mortality and not treatment-related. All surviving earthworms in the control and the treatment groups were normal in appearance and behavior. Earthworms showed no aversion to soils treated with the test substance. Since there was no treatment-related mortality of adult earthworms in the study, an LC50 value for mortality was not calculated.

There were no apparent effects upon earthworm weight in the treatment groups during the 28-day adult exposure period.
There were no statistically significant differences between mean final body weight, nor change in body weight, for the treatment groups when compared to the pooled control group means.

The juveniles collected from the control and treatment groups were normal in appearance and behavior. Only the mean number of juveniles produced in the 125 mg/kg dry soil group was statistically significantly different from the mean number of juveniles in the pooled control group with Dunnett’s Test (p<0.05). Based on scientific judgment this reduction was not considered to be due to be dose-responsive, since no other level showed a reduction in the number of juveniles produced.

Recoveries of test substance in artificial soil samples collected at Day 0 were in the range of 101.8 to 108.0% with an overall mean of 104.5%. Recoveries of test substance in artificial soil samples collected at Day 56 were in the range of 88.9 to 97.3% with an overall mean of 94.1%.

Results with reference substance (positive control):

Wildlife International conducted a reference toxicity test with carbendazim in 2006 to document that the earthworms being cultured were sensitive to a known toxicant. The LC50 value for the mortality of the adult earthworms exposed to carbendazim for 28 days was 7.149 mg a.i./kg dry soil, with a 95% confidence interval of 6.338 and 8.273 mg a.i./kg dry soil. There were effects upon adult earthworm weight at concentrations of 0.3 mg a.i./kg and in the 2, 4 and 8 mg a.i./kg groups.
The EC50 value for reproduction was 0.8914 mg a.i./kg dry soil, with a 95% confidence interval of 0.8416 and 0.9718 mg a.i./kg dry soil. The NOEC was 0.5 mg a.i./kg dry soil, and the LOEC was 1 mg a.i./kg dry soil, based on the numbers of juveniles produced.

Reported statistics and error estimates:

Replicate and treatment group means were determined from data collected. Mean numbers of juveniles produced, adult body weight, changes in adult body weights and adult mortality of the negative and solvent control groups were first compared with a t-test. The two control groups were not significantly different for these variables therefore the replicates of the two control groups were averaged together as a pooled control for comparison to treatment groups. Differences between the treatment groups and the pooled control group were evaluated to assess potential effects on fecundity, body weight, changes in body weight and adult mortality. Initial and final adult body weight, adult mortality and the number of juvenile worms were determined for each replicate. Treatment group means were calculated and compared to the pooled control group means using Dunnett’s Test in order to determine which group(s), differed significantly from the pooled control at the 0.05 level of significance. Prior to conducting Dunnett’ test, the data were tested for homogeneity of variance and normal distribution. Results of the statistical tests were used to help determine the NOEC and LOEC for body weight, adult mortality and production of juveniles. The NOEC was defined as the maximum test concentration at which there were no treatment-related reductions in adult body weight, adult mortality or a reduction in the number of offspring. The LOEC was defined as the lowest test concentration that showed effects for the same categories. Statistical calculations were made using a personal computer and commercially available software. The EC/LC50 is defined as the concentration of test substance that caused a 50% reduction relative to the pooled control group for a variable of concern. No EC/LC50 calculations were warranted for body weights, adult mortality or reproduction due to the lack of a 50% effect at any test level.

Validity criteria fulfilled:

yes

Conclusions:

There was no mortality of adult earthworms exposed to Naugalube 438L at 62.5, 125, 250, 500 and 1000 mg/kg dry soil for 28 days, other than one dead earthworm in the 1000 mg/kg dry soil group.
Based on body weight and survival data of adult earthworms, and reproduction, the NOEC was determined to be 1000 mg/kg dry soil, the highest concentration tested.

Description of key information

No toxic effects have been observed in studies with soil living organisms at the highest test concentration.

NOEC >= 1000 mg/kg DW

Key value for chemical safety assessment

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

1 000 mg/kg soil dw

Additional information

The toxicity of the test item to soil macroorganisms except arthropods was determined over a period of 28 days concerning mortality, body weight developement as well as 56 days concerning juvenile production according to OECD 222.

The test was conducted with earthworms (Eisenia fetida) with test concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight (DW).

No significant toxic effects were observed for all species at the highest test concentration.

A NOEC >= 1000 mg/kg DW was determined for all observed endpoints.