2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate

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:

11 October 2012 to 03 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was performed in accordance with ISO, Federal Biological Research Centre for Agriculture and Forestry (BBA) and OECD test guidelines in an accredited GLP laboratory.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

April 2004.

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Version / remarks:

July 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Biologische Bundesanstalt für Land- und Forstwirtschaft Bundesrepublik Deutschland: Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren

Version / remarks:

Part VI, 2-2: „Auswirkungen von Pflanzenschutzmitteln auf die Reproduktion und das Wachstum von Eisenia fetida/Eisenia andrei“, January 1994.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No other information provided

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): - Sphagnum peat (shredded, sieved through 2 mm sieve): 10%- Kaolin clay: 20%content of Al2O3: 34.2% content of kaolinite: approx. 87%- Sand (Sihelco 36): 69%Size analysis: 0.25 mm 1% 0.18 mm 7% 0.125 mm 80% 0.09 mm 11% <0.09 mm 1% - Calcium carbonate (CaCO3): about 0.6% (for the adjustment to pH 6.0 ± 0.5) - Food (dried organic manure): 1%The air dry components of the artificial soil (without calcium carbonate and food) were intensively mixed in a batch of 20 kg (dry weight) in a laboratory mixer to prepare a substrate mixture with the composition of peat, kaolinite clay, and sand as mentioned above. For each test vessel, 506.5 g of the prepared air dry artificial soil (corresponding to 500 g dry weight and 650 g fresh weight) was mixed with 3.1 g calcium carbonate and 5 g food in a laboratory mixer. During mixing, 100 mL of purified water, containing the desired concentration of the test item was added. Finally, 43.5 mL of purified water was added. Thus, adding in total 143.5 mL of purified water to the artificial soil, its moisture content was adjusted to 30%, corresponding to approximately 60% of its water holding capacity. The WHC amounted in average to 50.3 g water per 100 g dry soil. The positive control with Derosal, was applied as aqueous application solution analogously to the test item, using the same artificial soil.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The study was performed with earthworms of the species Eisenia fetida. The worms were obtained from a synchronized culture maintained at Blades Biological Ltd, Cowden / United Kingdom. During breeding and keeping, they were fed with suitable food (e.i., horse manure and potatoes). For one day prior to the test start, the test organisms were acclimatized to the artificial soil and test temperature. The individual body wet weight of the test organisms at the start of the test ranged from 310 to 595 mg (the worms were weighed individually at the test start of the study).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

Adult earthworms were exposed in treated soils for a period of four weeks. After this period, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks.

Test temperature:

Room temperature was in the range of 20 to 22 °C.

pH:

Between 6.0 - 6.3 at the start of the test and 6.0 - 6.1 by the end of the test.

Moisture:

Plastic boxes (length about 16.5 cm, width about 12 cm, height about 6.5 cm, area about 200 cm2) were used as test vessels. Each test vessel contained a defined amount of dry artificial soil, corresponding to a layer depth of approximately 5-6 cm. The test vessels were covered by transparent lids to prevent worms from escaping and to reduce evaporation during the test period. The lids were perforated to allow air exchange. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification. During the whole test period, they were kept in a temperature-controlled room, set at 20 ± 2 °C under a 16-hour light to 8-hour darkness photoperiod (light intensity within 706-776 Lux). For each treatment, four test vessels (replicates) with ten earthworms each were prepared. Thus, in total 40 test organisms were tested per treatment. However, for the control group, eight test vessels (replicates) with ten earthworms each were prepared, i.e. a total of 80 test organisms. To demonstrate satisfactory test conditions, a positive control with the reference item Derosal® with the active ingredient carbendazim was performed. Immediately after soil treatments, ten of the weighed test organisms were randomly placed on the soil surface of each test vessel.

Details on test conditions:

Plastic boxes (length about 16.5 cm, width about 12 cm, height about 6.5 cm, area about 200 cm2) were used as test vessels. Each test vessel contained a defined amount of dry artificial soil, corresponding to a layer depth of approximately 5-6 cm. The test vessels were covered by transparent lids to prevent worms from escaping and to reduce evaporation during the test period. The lids were perforated to allow air exchange. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification. During the whole test period, they were kept in a temperature-controlled room, set at 20 ± 2 °C under a 16-hour light to 8-hour darkness photoperiod (light intensity within 706-776 Lux). For each treatment, four test vessels (replicates) with ten earthworms each were prepared. Thus, in total 40 test organisms were tested per treatment. However, for the control group, eight test vessels (replicates) with ten earthworms each were prepared, i.e. a total of 80 test organisms. To demonstrate satisfactory test conditions, a positive control with the reference item Derosal® with the active ingredient carbendazim was performed. Immediately after soil treatments, ten of the weighed test organisms were randomly placed on the soil surface of each test vessel.

Nominal and measured concentrations:

The soil moisture content was sufficiently constant (29% at test start and 35-36% at test termination after 8 weeks)

Reference substance (positive control):

yes

Remarks:

see "Any other information on materials and methods incl. tables" for more details

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: survival, growth and reproduction

Key result

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: survival, growth and reproduction

Details on results:

Survival of Adults and Adverse Effects In the control group, the mortality rate of the adult test organisms after four weeks was 6.3%. Also at all test item concentrations up to and including 1000 mg/kg dry soil, the mortality rates did not exceed 7.5%. These mortality rates were not estimated as a significant toxic effect, because according to the test guidelines, a mortality rate up to 10% is considered as natural, and additionally, no concentration-effect relationship was observed. Thus, the survival rate of the earthworm Eisenia fetida after four weeks of exposure was not affected up to and including the highest concentration of 1000 mg 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) per kg dry soil. Moreover, no adverse effects were observed in the adult test organisms at any test concentration.Growth of Adults The mean body weight of the adult worms in the control had increased during the 4-week exposure period on average by 95 mg per worm (23% of the mean initial weight). At all test concentrations of the test item, the mean increase in body weight was statistically not significantly lower than in the control (results of a Dunnett t-test, one-sided smaller, α = 0.05). The results for the growth of the test organisms correspond to the observation that no prominent difference in food consumption was determined between the control and all treatments up to and including the highest test concentration of 1000 mg 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) per kg dry soil.Reproduction In the control, an average of 90 juvenile worms per test vessel were found (corresponding to a mean reproduction rate of 9.6 juveniles per surviving adult). The coefficient of variance of the reproduction rate per test vessel in the control was 27%. Thus, the validity criteria of the test guidelines (at least 30 juveniles per test vessel, coefficient of variance of the reproduction ≤30%) were fulfilled.At all test item concentrations up to and including 1000 mg/kg dry soil, the mean reproduction rates per vessel corresponded to 74-112% of the control value without a concentration-effect relationship. According to the results of a Dunnett t-test (one-sided smaller, α = 0.05), the mean reproduction rates were not statistically significantly reduced up to and including the highest test concentration of 1000 mg 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) per kg dry soil.NOEC and LOEC Taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) without significant adverse effects (NOEC) on Eisenia fetida after the test period was determined to be at least 1000 mg/kg dry soil. The NOEC might even be higher, but concentrations in excess of 1000 mg/kg dry soil were not tested. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.Soil Moisture, pH, and Temperature The soil moisture content was sufficiently constant (29% at test start and 35-36% at test termination after 8 weeks). The pH value in the test replicates was between 6.0 and 6.3 at the test start and between 6.0 and 6.1 at the end of the test. During the entire test period, the room temperature was in the range of 20 to 22 °C.

Results with reference substance (positive control):

Reference Item DerosalAt the single concentration of the reference item Derosal (8.33 mg formulation/kg dry soil, i.e. 5 mg a.i./kg dry soil), the mortality rate of the adult worms was 15%. The mean decrease of 2% in body wet weight of the earthworms in the positive control during the exposure period was significantly different to the control (results of a Student-t-test, one-sided smaller, α = 0.05). The reproduction rate of Eisenia fetida in the positive control was completely inhibited. Therefore, the results of the positive control demonstrated satisfactory test conditions.

Reported statistics and error estimates:

None specified

Validity criteria fulfilled:

yes

Conclusions:

Taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) without significant adverse effects on Eisenia fetida (NOEC) after the test period was determined to be the highest tested concentration of 1000 mg/kg dry soil. The reproduction rate of Eisenia fetida in the positive control was completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions. The validity criteria were fulfilled.

Executive summary:

The toxic effects of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guidelines for Testing Chemicals, Test No. 222 (April 2004), the International Standards ISO 11268-2: 1998(E) and the BBA Guideline “Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren“ Part VI, 2-2, January 1994.

 

2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) was homogeneously mixed into artificial soil at the following test concentrations:

 

10, 32, 100, 320 and 1000 mg per kg dry soil.

 

Additionally, a control treatment (without test item) and a reference item treatment (Derosal®8.33 mg/kg dry soil) were tested in parallel.

 

Adult earthworms were exposed in treated soils for a period of four weeks. After this period, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The

reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks.

 

Summary of effects on earthworms exposed to the test item:

Parameters	Control	2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) (mg/kg dry soil)					Derosal® (mg/kg dry soil)
Mortality (%) after a 4-week exposure	6.25	5	2.5	2.5	2.5	7.5	15
Mean change in body fresh weight (%) after a 4-week exposure	23	34	28	33	33	28	-2
Mean reproduction rate (juveniles per replicate)	90	100	87	90	101	67	0
Mean reproduction rate (juveniles per replicate)	100	111	96	100	112	74	0.0

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) without significant adverse effects onEisenia fetida(NOEC) after the test period was determined to be the highest tested concentration of 1000 mg/kg dry soil.

The reproduction rate of Eisenia fetida in the positive control was completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions.

Description of key information

Eisenia fetida (NOEC) after the test period was determined to be the highest tested concentration of 1000 mg/kg dry soil.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw

Additional information

The toxic effects of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guidelines for Testing Chemicals, Test No. 222 (April 2004), the International Standards ISO 11268-2: 1998(E) and the BBA Guideline “Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren“ Part VI, 2-2, January 1994.

 

Taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) without significant adverse effects on Eisenia fetida (NOEC) after the test period was determined to be the highest tested concentration of 1000 mg/kg dry soil.

In conclusion based on the information provided by the study it is appropriate to assume that 2,2-dimethylpropane-1,3-diyl 2-ethylhexanoate (CAS 28510-23-8) will not result in terrestrial toxicity via terrestrial bioccumulation and as such secondary poisoning to wild animals/invertebrates and indirect toxicity to humans is not likely to occur.