Phosphoric acid, mono- and di-C16-18-alkyl esters

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:

14 September 2017 to 10 September 2019

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))

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Physical State/Appearance: White waxy solid
Purity: 100%
Storage Conditions: Room temperature in the dark (15 - 25 °C)

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

The test material was weighed separately for each rate of application and dispersed in 100 mL of acetone by warming to 60°C in a water bath. This was poured over 500 g dry soil and the solvent was allowed to evaporate in a fume hood before addition to a 1700 g dry weight equivalent of moist soil. Reverse osmosis (RO) water (374.38 mL) was then added to bring the water content to 55% of MWHC. The soil and water were mixed into the moist soil using a hand held electric mixer.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Earthworms (Eisenia fetida) were purchased from a reputable supplier of laboratory livestock. Prior to study start the worms were maintained in animal manure and fed regularly. Details of suppliers are recorded only in the raw data to maintain supplier confidentiality.

Selection of adult worms was based primarily on individual weights falling within the specified weight range of 300 – 600 mg at the start of the treatment period. Prior to treatment, the batch of worms was inspected for reproductive maturity, i.e. production of egg cocoons or presence of a clitellum, the presence of which confirmed suitability for use.

Approximately 24 hours prior to study start adult worms were transferred to untreated artificial soil.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

28 d

Test temperature:

18.9 – 21.9 °C

pH:

6.7 - 6.8

Moisture:

43 to 49% MWHC

Details on test conditions:

Tests were conducted in plastic containers measuring approximately 11 x 17 x 5 cm, each covered with a tight fitting lid. After treatment, a weight equivalent to 500 g dry soil at 55% of the maximum water holding capacity was transferred into each of eight replicate containers for the water and solvent controls, four replicate containers for each treatment rate and eight for the positive control. Containers were labeled with the name of the study director, study number, test substance and rate of application, replicate number and date of application. The replicate groups of ten worms were then allocated randomly to each treatment using a bodyweight stratification procedure with the aid of a random number table.

An artificial soil (OECD 207) of the following composition was used as the test medium:

Industrial quartz sand 70% w/w
Kaolin clay 20% w/w
Peat 10% w/w

pH was adjusted to 6.0 ± 0.5 using calcium carbonate.

Untreated soil prior to treatment pH 6.5

The worms were maintained in a room which was designed to provide a satisfactory range of environmental conditions for the species. Minimum and maximum ambient temperatures and light intensity were recorded daily. The lights were controlled by a time switch set to provide a 16-hour light: 8-hour dark photoperiod.

On Day 28 of the study the soil was removed from the containers and the numbers and replicate weights of live adults were recorded. The soil was returned to the containers for the juvenile development phase. The juvenile worms were fed by carefully mixing 5 g of food (dried rabbit manure) into the substrate of each container. Juvenile worms were not fed further during the four-week rearing period.

Nominal and measured concentrations:

16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg active ingredient (a.i.)/kg dry soil

Reference substance (positive control):

yes

Remarks:

Treated with Ringer (active ingredient carbendazim)

Key result

Duration:

28 d

Dose descriptor:

EC50

Basis for effect:

other: bodyweight

Remarks on result:

not determinable

Remarks:

The EC50 for the mean body weight of the adult earthworms at Day 28 could not be estimated because there was not a dose-response relationship.

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: bodyweight

Key result

Duration:

56 d

Dose descriptor:

EC50

Basis for effect:

other: number of juveniles

Remarks on result:

not determinable

Remarks:

The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship.

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: number of juveniles

Details on results:

No adult mortality was recorded at any test material treatment rates. No adult mortality was recorded in the water control, solvent control or in the positive control group.

There were no statistically significant differences in mean adult bodyweights between the solvent control group and test material treatment groups.

The coefficient of variation of the number of juveniles in the solvent control group was 5.05%.

No statistically significant differences in the number of juveniles on Day 56 were identified between the test material treatment groups and the solvent control.

There were no statistically significant differences between the numbers of unemerged cocoons between the test material treatment groups and the solvent control.

Results with reference substance (positive control):

The application of carbendazim at 5 mg a.i./kg dry soil resulted in significant and unequivocal toxic effects (reduction in growth and reproduction) that were within the acceptable range for this toxic reference item.

Reported statistics and error estimates:

Statistical analysis was carried out using SAS 9.1.3 (SAS Institute 2002). See tables for further information.

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for the mean bodyweight at Day 28 was 1000 mg a.i./kg dry soil.

The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.

Executive summary:

A study was performed to determine the effects of the test material on the reproduction and growth of the earthworm,Eisenia fetida. 

A definitive study was conducted at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg active ingredient (a.i.)/kg dry soil. 

Eleven groups of worms were allocated to the study. Eight groups of 40 worms each were treated with the test material at a rate of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 or 1000 a.i./kg dry soil. Water and solvent control groups of 80 worms, were maintained in untreated soil to act as negative controls. A positive control group of 80 worms was treated with Ringer (active ingredient: carbendazim) at 5 mg a.i./kg dry soil. Adult worms were removed from the soil four weeks after treatment and the juvenile worms reared for a further four weeks.

No adult mortality was recorded at any test material treatment rates. No adult mortality was recorded in the water control, solvent control or in the positive control group. As there was no adult mortality at Day 28 in the treatment groups, no statistical analysis could be performed on Day 28 and the LC₅₀was therefore estimated to be >1000 mg a.i./kg dry soil. The No Observed Effect Concentration (NOEC) for adult mortality on Day 28 was 1000 mg a.i./kg dry soil. 

There were no statistically significant differences in mean adult bodyweights between the solvent control group and the test material treatment groups. The EC₅₀for the mean body weight of the adult earthworms at Day 28 could not be estimated because there was not a dose-response relationship. The NOEC for the mean bodyweight at Day 28 was 1000 mg a.i./kg dry soil.

The 5 mg a.i./kg dry soil carbendazim treated group had significantly lower body weight than the water control group.

The coefficient of variation of the number of juveniles in the solvent control group was 5.05%.

No statistically significant differences in the number of juveniles on Day 56 were identified between the test material treatment groups and the solvent control. The EC₅₀for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship. The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.

The 5  mg a.i./kg dry soil carbendazim treated group had significantly fewer juveniles than the water control group.

There were no statistically significant differences between the numbers of unemerged cocoons between the test material treatment groups and the solvent control. 

The study was considered valid as there was =10% adult mortality at four weeks and =30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction = 30%. In addition, the application of carbendazim at 5 mg a.i./kg dry soil resulted in significant and unequivocal toxic effects (reduction in growth and reproduction) that were within the acceptable range for this toxic reference item.

Description of key information

The NOEC for the mean bodyweight at Day 28 was 1000 mg a.i./kg dry soil.

The NOEC for the number of juvenile worms on Day 56 was 1000 mg a.i./kg dry soil.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw