Reaction products of C16-18 (even numbered), C18 unsaturated alkylamines with C10-13 alkylbenzenesulfonic acid

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:

February 2020 - September 2020

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 temperature in the definitive test rose to above the maximum temperature of 22°C to 24.6°C. As the validity criteria was met these deviations in temperature were not deemed to have effected the study.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The test item was weighed separately for each rate of application and dispersed in 100 mL of acetone. 100 mls was poured over 500 g dry soil and the solvent was allowed to evaporate in a fume hood before addition to a 2958.34 g of moist soil (equivalent to 2200 g dry soil). Reverse osmosis (RO) water (422.76 mL) was then added to bring the water content to 50% of MWHC. The dry soil and water were mixed into the moist soil using a hand held electric mixer.

- Controls: For the water control 807.0 mL RO water was mixed into a 3886.16 g of moist soil (equivalent to 3245.8 g dry soil) to which 954.5 g dry soil had been added. The mixing procedure employed was the same as used for the test item treatments. For the solvent control 190.9 mL of acetone was poured over 954.5 g of dry soil and allowed to evaporate. This was mixed into 3886.16 g of moist soil (equivalent to 3245.8 g dry soil) and 807.0 mL RO water added.

- Chemical name of vehicle (organic solvent): acetone

- Concentration of vehicle in test medium : For more details, see "Treatment table" in attached justification

- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida
- Source: Bias Labs, Fife, United Kingdom
- Age at test initiation (mean and range, SD): The worms are from a cohort of cocoons laid 17 September to 1st October 2019 and are therefore confirmed to be between 2 months and 1 year old.
- Weight at test initiation (mean and range, SD): 300 – 600 mg

ACCLIMATION
- Acclimation period: Prior to the start of the study, worms will be acclimatized to untreated soil for at least 24 hours, during which worms will be fed the same food as used in the test.
- Acclimation conditions (same as test ): yes

FEEDING CONDITION DURING TEST
Dried rabbit manure was used as food. One day after application of the test item, 5 g of dried manure was uniformly distributed onto the surface of the soil in each test container and wetted with 5 mL of reverse osmosis water. Worms at each treatment rate were fed weekly in a similar way if two or more replicates per rate were assessed as having consumed 50% or more of the food.

After four weeks (after removal of adult worms), the juvenile worms will be fed by carefully mixing 5 g of food into the substrate for each container. Juvenile worms will not be fed further during the four-week rearing period.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Post exposure observation period:

none

Test temperature:

In the definitive test the temperature ranged from 18.9 – 24.6°C. The average temperature ranged from 19.8 – 22.6°C.

pH:

A sample was taken from the control and each treatment rate for pH sampling at the study start and at Day 56.

pH Min : 5.7 - Max : 6.4

For more details, see table "Test Soil pH" in attached justification.

Moisture:

Prior to the start of the study, the batch of soil was sampled to assess the water holding capacity. The maximum water holding capacity (MWHC) of the soil was determined before treatment as 68.94%. A moisture content equivalent to 50% of the MWHC was selected as providing suitable conditions for earthworm development. The volume required to achieve 50% of the maximum water holding capacity was calculated as 422.76 mL water/2200 g dry ISO earthworm soil. No pre-moistening of the soil was required as the water content was already >25% of the MWHC.

Moisture content was recorded at Day 0 and Day 56. A container of soil was maintained under identical conditions to the test containers and was used as a guide for maintaining the moisture content of the test containers throughout the study. At termination, group replicate mean moisture content values were found to be 31.7 to 34.7%, equivalent to 46.0 – 50.4% MWHC.

Details on test conditions:

TEST SYSTEM
- Test container : Tests were conducted in plastic containers measuring approximately 11 x 17 x 5 cm.
- Amount of soil or substrate: 750 g/replicate
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: /
- Pesticide use history at the collection site: /
- Collection procedures: /
- Sampling depth (cm): /
- Soil texture (if natural soil): NA
- % sand:
- % silt:
- % clay:
- Soil taxonomic classification: NA
- Soil classification system: Artificial
- Composition (if artificial substrate): A batch of artificial soil was prepared at Covance in the Terrestrial Ecotoxicology department. The soil used as the test medium was prepared with the following composition, as specified in OECD Guideline 222:
Industrial quartz sand (fine sand, 50% particle size of 0.05 to 0.2 mm) : 70% w/w
Kaolin clay (>30%) : 20% w/w
Peat : 10 % w/w
pH was adjusted to 6.0 ± 0.5 using calcium carbonate
- Organic carbon (%): /
- Maximum water holding capacity (in % dry weigth): The maximum water holding capacity (MWHC) of the soil in the definitive test was determined before treatment as 68.94%. (Then ajusted see details below)
- CEC: /
- Pretreatment of soil: Untreated soil prior to treatment pH 5.8 for the range finder and pH 6.0 for the definitive
- Storage (condition, duration): /
- Stability and homogeneity of test material in the medium: /

OTHER TEST CONDITIONS
- Photoperiod: 16-hour light: 8-hour dark photoperiod
- Light intensity: 410 – 739 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality, Bodyweights, Juvenile Worms

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1,8 (definitive test)
- Justification for using less concentrations than requested by guideline: none
- Range finding study : yes
- Test concentrations: 0,1 - 1 - 10 - 100 -1000 mg a.i./ kg dry soil
- Results used to determine the conditions for the definitive study: There was 5% mortality in the water control group and no mortality in the solvent control or at Amines tallow alkyl dodecylbenzenesulfonates treatment rates of 0.1, 10, 100 and 1000 mg a.i./kg dry soil a 5% mortality was observd at 1.0 mg a.i./kg dry soil. The mortality in the controls did not exceed 10 per cent at the end of the test. There was a slight significant treatment related difference in bodyweight at 100 and 1000 mg a.i./kg dry soil on Day 14. The 14-day LC50 value was estimated to be 1000 mg a.i./kg dry soil.

Nominal and measured concentrations:

Eleven treatments were employed in the study. These consisted of:
i) Water control.
ii) Solvent control.
iii) Amines tallow alkyl dodecylbenzenesulfonates applied at 16.3 mg a.i./kg dry soil.
iv) Amines tallow alkyl dodecylbenzenesulfonates applied at 29.4 mg a.i./kg dry soil.
v) Amines tallow alkyl dodecylbenzenesulfonates applied at 52.9 mg a.i./kg dry soil.
vi) Amines tallow alkyl dodecylbenzenesulfonates applied at 95.3 mg a.i./kg dry soil.
vii) Amines tallow alkyl dodecylbenzenesulfonates applied at 171.5 mg a.i./kg dry soil.
viii) Amines tallow alkyl dodecylbenzenesulfonates applied at 308.6 mg a.i./kg dry soil.
ix) Amines tallow alkyl dodecylbenzenesulfonates applied at 555.6 mg a.i./kg dry soil.
x) Amines tallow alkyl dodecylbenzenesulfonates applied at 1000 mg a.i./kg dry soil.
xi) Ringer (carbendazim) applied at 5 mg a.i./kg dry soil.

Reference substance (positive control):

yes

Remarks:

Ringer (carbendazim)

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

95.3 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

number of juvenile worms

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

numbers of unemerged cocoons and number of juveniles

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

Adults

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks:

Adults

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

Adult bodyweight

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Remarks:

Adult bodyweight

Details on results:

- Mortality at end of exposure period: Adult mortality of 0% was recorded in the 16.3, 52.9, 95.3, 308.6, 555.6 and 1000 mg a.i./kg dry soil and 2.5% mortality (not significant) in the 29.4 and 171.5 mg a.i./kg dry soil Amines tallow alkyl dodecylbenzenesulfonates treatment rates.
- Total mass of adults at beginning of test: 340 - 342 mg/earthworm (Day 0)
- Changes in body weight of live adults (% of initial weight) at end of exposure period: There was no statistically significant adverse effect on adult bodyweight at any Amines tallow alkyl dodecylbenzenesulfonates rates in comparison to the solvent control.
- No. of offspring produced: Statistically significant differences in the number of juveniles were recorded on Day 56 at the Amines tallow alkyl dodecylbenzenesulfonates groups of 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil compared against the solvent control.
- No. of unhatched cocoons: There were no statistically significant differences between the numbers of unemerged cocoons between test item treatment groups and the solvent control.
- Morphological abnormalities: No pathological signs were observed.
- Behavioural abnormalities: No behavioural signs were observed.
- Other biological observations: /

Results with reference substance (positive control):

- Results with reference substance valid : yes
- Relevant effect levels:
Adult bodyweight : The 5 mg a.i./kg dry soil carbendazim treated group had significantly lower body weight than the water control group.
Juveniles worms : The 5 mg a.i./kg dry soil carbendazim treated group had significantly fewer juveniles than the water control group. 100% reproduction inhibition was observed at 5 mg a.i./kg dry soil therefore the TG OECD 222 validity criteria for the reference control was fulfilled.

Reported statistics and error estimates:

Statistical analysis was carried out using SAS 9.1.3, 9.4 (SAS Institute 2002) and Quasar 1.5 (Quasar 1.5 2017) (Annex 2).
Major Computerized Systems : The IT systems listed below were used on this study.
Pristima: Test item management
REES: Environmental monitoring system (temperature only)
SAS 9.1.3, 9.4 and Quasar 1.5: Statistics

Table1          % Adult Mortality at Day 28 and Treatment Mean Bodyweights (mg)

Treatment	(mg a.i./kg dry soil)	% Mortality	p-value	Day of study			(%) Change^(b)	p-value
				Day 0	Day 28	Day 28^(a)
Water control	0	1.25	-	343	641	640	+86.9	-
Solvent control	0	2.5	0.500	341	619	619	+81.5	0.115
Test item	16.3	0	>0.999	341	625	625	+83.3	1.000
	29.4	2.5	0.707	341	653	653	+91.5	0.243
	52.9	0	>0.999	341	640	640	+87.7	0.770
	95.3	0	>0.999	341	631	630	+85.0	0.992
	171.5	2.5	0.707	341	638	639	+87.1	0.819
	308.6	0	>0.999	341	628	629	+84.2	0.997
	555.6	0	>0.999	340	604	605	+77.6	0.954
	1000	0	>0.999	342	656	656	+91.8	0.170
Carbendazim	5	0	>0.999	342	337	336	-1.8	<0.001***

For mortality all test item treatment rates were compared against the solvent control group and the carbendazim was compared against the water control using fishers exact test

For the bodyweight all test item treatment rates were compared against the solvent control group using Dunnett’s test and the carbendazim was compared against the water control using t-test

 ***p<0.001

- Not applicable, (a) adjusted mean weights, (b) calculated from non-adjusted means

Table2          Mean Number of Juvenile Worms per Treatment Replicate Day 56

Treatment	n	Mean	Percent reduction in reproduction	sd	p-value
Water control	8	371	0	13.2	0.326
Solvent control	8	362	-	19.8	-
Test Item 16.3 mg a.i./kg dry soil	4	353	2.5	37.6	0.868
Test Item 29.4 mg a.i./kg dry soil	4	353	2.5	34.0	0.868
Test Item 52.9 mg a.i./kg dry soil	4	313	13.5	57.9	0.226
Test Item 95.3 mg a.i./kg dry soil	4	335	7.5	49.7	0.226
Test Item 171.5 mg a.i./kg dry soil	4	274	24.3	56.3	0.015*
Test Item 308.6 mg a.i./kg dry soil	4	264	27.1	31.4	0.005**
Test Item 555.6 mg a.i./kg dry soil	4	273	24.6	11.3	0.005**
Test Item 1000 mg a.i./kg dry soil	4	284	21.6	56.8	0.005**
Carbendazim 5 mg a.i./kg dry soil	8	0	100.0	0.4	<0.001***
Water control CV(%)					3.56
Solvent control CV(%)					5.49

n           Number of replicates
sd          Standard deviation

-            Not applicable

CV        Coefficient of variation

All test item treatment rates were compared against the solvent control group using Shirley’s test and the carbendazim was compared against the water control using Wilcoxon’s sum of ranks test

*p<0.05, **p<0.01,***p<0.001

Table 3   Number of un-emerged cocoons on Day 56

Treatment group	Day 56
	n	Mean	p-value
Statistics test:		KW
Water control	8	0.75	1.000
Solvent control	8	0.50	-
Test Item 16.3 mg a.i./kg dry soil	4	0.25	-
Test Item 29.4 mg a.i./kg dry soil	4	0.75	-
Test Item 52.9 mg a.i./kg dry soil	4	0.00	-
Test Item 95.3 mg a.i./kg dry soil	4	0.00	-
Test Item 171.5 mg a.i./kg dry soil	4	0.25	-
Test Item 308.6 mg a.i./kg dry soil	4	0.25	-
Test Item 555.6 mg a.i./kg dry soil	4	0.00	-
Test Item 1000 mg a.i./kg dry soil	4	1.00	-
Carbendazim 5 mg a.i./kg dry soil	8	3.88	0.010
Overall testp-value			0.704

All test item treatment rates were compared against the solvent control group using Kruskal-Wallis test and the carbendazim was compared against the water control using Wilcoxon’s sum of ranks test

- not applicable

n = Number of replicates

Validity criteria fulfilled:

yes

Remarks:

Each replicate (containing ten adults) to have produced >= 30 juveniles by the end of the test. The coefficient of variation of reproduction to be <= 30%. Adult mortality over the initial four weeks to be <= 10%.

Conclusions:

The LC50 for adult mortality could not be estimated due to a lack of dose response it was therefore concluded to be >1000 mg a.i./kg dry soil. The NOEC for adult mortality on Day 28 was 1000 mg a.i./kg dry soil.

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 it was therefore concluded to be >1000 mg a.i./kg dry soil. The NOEC for the mean bodyweight at Day 28 was 1000 mg a.i./kg dry soil.

The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship. The IC10 for the number of juveniles on Day 56 was estimated as 98 mg a.i/kg dry soil (95% confidence interval of 27 to 362 mg a.i/kg dry soil).The NOEC for the number of juvenile worms on Day 56 was 95.3 mg a.i./kg dry soil. There were no statistically significant differences between the solvent control and the Amines tallow alkyl dodecylbenzenesulfonates treatment groups in the number of un-emerged cocoons.

Executive summary:

A study according GLP and TG OECD 222 was performed to determine the effects of the registered substance on the reproduction and growth of the earthworm, Eisenia fetida. The test item was applied to the artificial soil to give an indication of the effects on the test organism following exposure in the field. The data generated in this study met all of the following validity criteria of the TG OECD 222.

 

A preliminary range finder test was conducted at treatment rates of 0.1, 1, 10, 100 and 1000 mg a.i./kg dry soil to determine the acute toxicity of the test item to the earthworm Eisenia fetida in an artificial soil under laboratory conditions and to ensure selection of appropriate treatment rates for the reproduction test.  Water and solvent controls were also included.

 

There was 5% mortality in the water control group and no mortality in the solvent control or at test item treatment rates of 0.1, 10, 100 and 1000 mg a.i./kg dry soil a 5% mortality was observd at 1.0 mg a.i./kg dry soil.  There was a slight significant treatment related difference in bodyweight at 100 and 1000 mg a.i./kg dry soil on Day 14.  The 14-day LC50 value was estimated to be 1000 mg a.i./kg dry soil. Based on these results the definitive test was conducted which was comprised of a water control, solvent control, test item treatments applied at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil and toxic reference Ringer (carbendazim), applied at 5 mg a.i./kg dry soil.

 

Adult mortality

Mortality in the water control was 1.25% and in the solvent controls was 2.5% and no mortality was recorded in the toxic reference treatment.

 

Adult mortality of 0% was recorded in the 16.3, 52.9, 95.3, 308.6, 555.6 and 1000 mg a.i./kg dry soil and 2.5% mortality (not statisticaly significant) in the 29.4 and 171.5 mg a.i./kg dry soil treatment rates.

 

The LC50 for mortality could not be estimated due to a lack of dose response and was therefore concluded to be >1000 mg a.i./kg dry soil.

 

The No Observed Effect Concentration (NOEC) for adult mortality at Day 28 was 1000 mg a.i./kg dry soil.

 

Adult bodyweight

There were no statistically significant differences in mean bodyweights between the solvent control group and test item treatment groups.  The 5 mg a.i./kg dry soil carbendazim treated group had significantly lower body weight than the water control group.

 

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.

 

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

 

Juvenile worms

The coefficient of variation of the number of juveniles in the water control group was 3.56% and in the solvent control group 5.49%.

Statistically significant differences in the number of juveniles were recorded on Day 56 at the test item treatment groups of 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil compared against the solvent control. The 5 mg a.i./kg dry soil carbendazim treated group had significantly fewer juveniles than the water control group. 100% reproduction inhibition was observed at 5 mg a.i./kg dry soil therefore the TG OECD 222 validity criteria for the reference control was fulfilled.

The EC50 for the number of juveniles on Day 56 could not be estimated because there was not a dose-response relationship and was therefore concluded to be >1000 mg a.i./kg dry soil.

The NOEC for the number of juvenile worms on Day 56 was 95.3 mg a.i./kg dry soil.

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

 

The study was considered valid as there was less than 10% adult mortality at four weeks and more than 30 juveniles had been produced in each water and solvent control replicate by the end of the test with the coefficient of variation of reproduction less than 30%. In addition, application of the toxic reference Ringer at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.

Description of key information

According the OECD 222 and GLP study, the chronic toxicity of the registered substance (EC 701-164 -2) on earthworms is NOEC = 95.3 mg a.i/kg dry soil (number of juvenile worms on Day 56).

Key value for chemical safety assessment

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

95.3 mg/kg soil dw

Additional information

Long term toxicity on earthworms (OECD 222, GLP):

A study was performed to determine the effects of the test item (EC 701-164-2) on the reproduction and growth of the earthworm, Eisenia fetida. The study is considered valid as all validity criteria are fulfilled according TG OECD 222. The test item was mixed with the artificial soil to give an indication of the effects on the test organism following exposure in the field.

 

The definitive test was conducted which was comprised of a water control, solvent control, Amines tallow alkyl dodecylbenzenesulfonates treatments applied at 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg a.i./kg dry soil and toxic reference Ringer (carbendazim), applied at 5 mg a.i./kg dry soil.

 

Endpoints for adult mortality was demonstrated to be > and = 1000 mg a.i./kg dry soil respectively. The EC50 for the number of juveniles on Day 56 could not be estimated because there was not dose-response relationship and was therefore concluded to be >1000 mg a.i./kg dry soil. The NOEC for the number of juvenile worms on Day 56 was 95.3 mg a.i./kg dry soil. There were no statistically significant differences between the numbers of unemerged cocoons between test item treatment groups and the solvent control.

 

The study was considered valid as there was less than 10% adult mortality at four weeks and more than 30 juveniles had been produced in each water and solvent control replicate by the end of the test with the coefficient of variation of reproduction was less than 30%. In addition, application of the toxic reference Ringer at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.