Alcohols, C20-22, reaction products with phosphorus oxide (P2O5)

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 October 2019 - 15 January 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))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

White Flakes

Analytical monitoring:

no

Details on sampling:

Concentrations: 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.
The number of surviving juvenile worms in each replicate was determined on Day 56. Worms visible on the soil were removed and counted. Test containers were then removed from the water bath and emptied onto a tray. As soon as possible after removal from the water bath, a sample of soil was taken from each test container, checked to ensure it contained no worms and weighed accurately, before being dried and used to determine the moisture content at termination.
Moisture content was recorded at Day 1 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.
A sample was taken from the control and each treatment rate for pH sampling at the study start and completion.

Vehicle:

no

Details on preparation and application of test substrate:

The test substance was weighed separately for each rate of application and dispersed in 100 mL of acetone. This was poured over 500 g dry soil and the solvent was allowed to evaporate in a fume hood before addition to a 1964.69 g dry weight equivalent of moist soil. Reverse osmosis (RO) water (326.89 mL) was then added to bring the water content to 50% of maximum water holding capacity (MWHC). The dry soil and water were mixed into the moist soil using a hand held electric mixer.

For the water control 624.06 mL RO water was mixed into a 3750.77 g soil dry weight equivalent of moist soil to which 954.55 g dry soil had been added.
For the solvent control 191 mL of acetone was poured over 954.55 g of dry soil and allowed to evaporate. This was mixed into 3750.77 g soil dry weight equivalent of moist soil and 624.06 mL RO water added.
For the positive control group, 42 µL of Ringer (500 g/L carbendazim) was added to a final volume of 100 mL RO water to give a solution containing 0.21 mg a.i./mL. This was mixed into a 3750.77 g dry weight equivalent of moist soil to which 954.55 g of untreated dry soil had been added. A further 524.06 mL of RO water was added to bring the soil to 50% of the maximum water holding capacity.

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 50% 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. 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.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Earthworms (Eisenia fetida) and dried rabbit manure were purchased from a reputable supplier of laboratory livestock. Prior to study start the worms were maintained in animal manure and fed regularly.

Selection of adult worms for the study 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 also inspected for reproductive maturity. The worms supplied had clitella the presence of which confirmed maturity and suitability for use.
Approximately 24 hours prior to the 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

Remarks:

The test item was applied to artificial soil in order to give an indication of the effects on the test organism following field application.

Post exposure observation period:

On Day 28 of the study the soil was removed from the containers and the numbers and replicate weights of live adults were recorded. No behavioural and/or pathological signs were observed. 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.

Test temperature:

19.2 – 21.8 (degrees Centigrade)

pH:

pH was adjusted to 6.0 ± 0.5 using calcium carbonate (Untreated soil prior to treatment pH 5.6).
See table 1.

Moisture:

No pre-moistening of the soil was required as the water content was already >25% of the MWHC.

Details on test conditions:

TEST SYSTEM
Prior to application selected worms were rinsed in reverse osmosis water, blotted dry and individually weighed to ensure that all individual weights fell within the specified range (300 - 600 mg) before being reweighed in replicates of ten worms. 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 50% 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.
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. The quantity of water given was based on the amount of water loss from the moisture control box each week.

SOURCE AND PROPERTIES OF SUBSTRATE -
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

OTHER TEST CONDITIONS
The lights were controlled by a time switch set to provide a 16-hour light: 8-hour dark photoperiod. The light intensity range was 410 – 793 lux.

EFFECT PARAMETERS MEASURED:
Health and Mortality - On Day 28 of the study the soil was removed from the containers and the numbers and replicate weights of live adults were recorded. No behavioural and/or pathological signs were observed. 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.
Bodyweights - Surviving adult worms were rinsed in reverse osmosis water, blotted dry and weighed in replicate groups at the end of the four week exposure period. The adult worms were then humanely euthanized by freezing before being discarded as clinical waste.
Juvenile Worms - The number of surviving juvenile worms in each replicate was determined on Day 56. Test containers were placed in a water bath set to 60°C to encourage juvenile worms to come to the soil surface. Worms visible on the soil were removed and counted. Test containers were then removed from the water bath and emptied onto a tray. As soon as possible after removal from the water bath, a sample of soil was taken from each test container, checked to ensure it contained no worms and weighed accurately, before being dried and used to determine the moisture content at termination. The remaining soil from each container was searched carefully and any remaining worms were removed and counted.

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
Based on the results of the rangefinder test the following rates were selected for the definitive test:
i) Water control.
ii) Solvent control.
iii) Distilled acetalization product between glucose and C12/18 alcohol applied at 16.3 mg a.i./kg dry soil.
iv) Distilled acetalization product between glucose and C12/18 alcohol at 29.4 mg a.i./kg dry soil.
v) Distilled acetalization product between glucose and C12/18 alcohol at 52.9 mg a.i./kg dry soil.
vi) Distilled acetalization product between glucose and C12/18 alcohol applied at 95.3 mg a.i./kg dry soil.
vii) Distilled acetalization product between glucose and C12/18 alcohol applied at 171.5 mg a.i./kg dry soil.
viii) Distilled acetalization product between glucose and C12/18 alcohol applied at 308.6 mg a.i./kg dry soil.
ix) Distilled acetalization product between glucose and C12/18 alcohol applied at 555.6 mg a.i./kg dry soil.
x) Distilled acetalization product between glucose and C12/18 alcohol applied at 1000 mg a.i./kg dry soil.
xi) Carbendazim applied at 5 mg a.i./kg dry soil.

Nominal and measured concentrations:

not stated in report

Reference substance (positive control):

yes

Key result

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

not determinable

Remarks:

Estimated

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:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: bodyweight

Remarks on result:

not determinable

Remarks:

Estimated

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: bodyweight

Key result

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

Remarks:

could not be established because there was no dose-response relationship

Remarks on result:

not determinable

Remarks:

could not be established because there was no dose-response relationship

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- One adult earthworm was recorded as unaccounted for at 29.4 mg a.i./kg and no mortality was observed at any other treatment rate for Reaction products between C20/22 (even numbered) alcohol and phosphoric anhydride treatment rate. No mortality was recorded in the water control or solvent control. Mortality in the positive control group was 10%. (See table 2).
- There were no statistically significant adverse effects in mean bodyweights between the solvent control group and Reaction products between C20/22 ( even numbered) alcohol and phosphoric anhydride treatment groups. The 5 mg a.i./kg dry soil carbendazim treated group had significantly lower body weight than the water control group. (See table 2).

- Water control and solvent group productivity was acceptable (mean of 447 and 440 juveniles respectively per replicate). The coefficient of variation of the number of juveniles in the solvent control group was 6.50% and 6.01% in the water control.
No statistically significant differences in the number of juveniles were recorded on Day 56 between the treatment groups and the solvent control. The positive control group, treated with 5 mg a.i./kg dry soil carbendazim had significantly fewer juveniles than the water control group (p<0.001). (See table 3).

Results with reference substance (positive control):

Mortality of 10% was recorded in the toxic reference group.
Application of the toxic reference carbendazim at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.

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).

Table 2 - % Adult Mortality and Treatment Mean Bodyweights (mg)

Treatment	Treatment Rate (mg a.i./kg dry soil)		Adult Bodyweight
		% Mortality Day 28		Day of Study		Change (%)	Day of Study	pvalue
				Time 0 (mg)	Day 28 (mg)		Day 28^(a)(mg)
Water control	0	0		472	801	+70	801	-
Solvent control	0	0		474	793	+67	793	0.545
Reaction products between C20/22 ( even numbered) alcohol and phosphoric anhydride	16.3	0		474	801	+69	801	0.940
	29.4	2.5		473	771	+63	771	0.344
	52.9	0		474	779	+64	779	0.344
	95.3	0		474	794	+68	794	0.344
	171.5	0		470	760	+62	760	0.344
	308.6	0		473	787	+66	787	0.344
	555.6	0		473	795	+68	795	0.344
	1000	0		474	778	+64	778	0.258
Ringer (Carbendazim)	5	10		472	282	-40	282	<0.001***

***p<0.001

p values for bodyweight (adjusted means) are in comparison with the solvent control using Williams’ testand Ringer in comparison to the water control using t-test

 ^(a) Mean adjusted bodyweight

- Not applicable

Table 3 - Mean Number of Juvenile Worms per Treatment Replicate

Treatment	Treatment rate (mg a.i./kg dry soil)	Mean Number of juveniles	Day 56
			p value
Water control	0	447	-
Solvent control	0	440	0.646
Reaction products between C20/22 ( even numbered) alcohol and phosphoric anhydride	16.3	451	>0.999
	29.4	440	>0.999
	52.9	457	>0.999
	95.3	443	>0.999
	171.5	445	>0.999
	308.6	444	>0.999
	555.6	453	>0.999
	1000	445	>0.999
Ringer (Carbendazim)	5	0	<0.001***

 p values for the test item are in comparison with the solvent control using Williams test and positive control (Ringer) in comparison to the water control usingt-test

***p< 0.001

-Not applicable

CV (%) = 6.50% (coefficient of variation for the solvent control group)

CV (%) = 6.01% (coefficient of variation for the water control group)

Validity criteria fulfilled:

yes

Conclusions:

The LC50 could not be established due to a lack of effect and was therefore estimated to be >1000 mg a.i./kg dry soil. The NOEC for 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 established due to a lack of adverse effect. The EC50 was therefore estimated 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 established due to a lack of a adverse effect. The EC50 was therefore estimated to be >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.
The study was considered valid as adult mortality was ≤ 10% at four weeks and ≥ 30 juveniles were produced in each water control replicate by the end of the test, with the coefficient of variation for reproduction of ≤ 30%. In addition treatment with the toxic reference carbendazim at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects that were within the acceptable range for this toxic reference item.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has GLP and Quality assurance statements approved by China authorities

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The statements (GLP, Quality assurance) are part of the report

Analytical monitoring:

not required

Vehicle:

yes

Details on preparation and application of test substrate:

Since the solubility of test substance is lower than 1mg/L in water, the test substance was weighed with a balance and mixed with silica sand. The mixture was mixed thoroughly with basic test substrates and then with deionized water to obtain an even distribution of the test substance in the test substrates.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The study was performed with earthworms (Eisenia foetida). This is the species recommended in Ministry of Environmental Protection of China, the Guildelines for the Testing of Chemicals, 2004, 207 Earthworm, Acute Toxicity Test. Ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive database for this species make them suitable for acute toxicity testing.
The test was performed with Eisenia foetida which were purchased from Shanghai Yinquan Agriculture Development Co., Ltd. (ID: EF-MKT-100930). All newly acquired earthworms were quarantined and observered for 14 days prior to use in the test. Earthworms were bred in cow dung and kept under 20±2°C for 7 days prior to testing. At the start of the test, the used adult earthworms, which were originated from the same population, were with clitellum and body weight of 300 to 600 mg.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

7 d

Remarks:

A range-finding test were carried out at 10, 100 and 1000 mg/kg dry artificial soil weight as well as a blank control. 10 earthworms were used for each concentration of treatment and contro

Post exposure observation period:

Based on result of range-finding test, one treatment group which nominal concentration was 1000 mg/kg dry artificial soil weight and one control group were conducted in definitive test. 40 earthworms were used for both groups, divided into four replicates of ten earthworms each.

Test temperature:

between 20.2-21.4°C

pH:

6.84 (at the beginning of the test)

Moisture:

The moisture of artificial soil were from 35.9-38.7% dry soil weight

Details on test conditions:

1L glass breakers were used as the test vessels. The test substrate for each concentration was divided into four equal quantities of 750 g as determined by weight. Each portion was placed into a separate glass beaker and represented one replicate for exposing 10 earthworms at the same concentration.

The earthworms, which were washed quickly with surplus water absorbed on filter paper before use, were randomly separated into groups of 10. These groups were weighed and then randomly assigned to the beakers by placed on the test substrate surface. The beakers were covered with film to prevent the substrate drying and were kept in artificial climate chamber for 14 d.

Each beaker was checked for dead and observations recorded at 7 and 14 d after the beginning of the test. The test substrates were spreaded on glass plate. The earthworms were examined and their reaction to a gentle mechanical stimulus were tested. Earthworms were considered dead if they did not respond to a gentle mechanical stimulus. Missing earthworms were also considered dead. Any adverse effects were recorded. When the examinaton was made at 7 d, the beaker was refilled with the substrate and the surviving earthworms were replaced on the same test substrate surface.

The moisture and temperature of the test substrates were measured at the beginning and the end of the test in a beaker of control group.
pH of the test substrates was measured at the beginning of the test in control group.

Nominal and measured concentrations:

The nominal concentration was 1000 mg/kg

Key result

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

In range-finding test, no mortality was observed in all three treatment groups

Mortalities in control group and treatment group were 0 on 7th and 14th day

The 7 days and 14 days LC₅₀value of the test substance toEisenia foetidawere higher than 1000 mg/kg dry soil weight

At the end of the test, the average body weight of the surviving earthworms in control group was significantly decrease(p<0.05). However, there were no significant differents (p>0.05) in the average body weight of the surviving earthworms between control group and treatment group

Conclusions:

Expressed as the nominal concentration of the test substance, the lethal effect of SENSANOV WR (SPQP20101202-0165) on Eisenia foetida is as following:
The 7 days LC50 value was higher than 1000 mg/kg dry soil weight.
The 14 days LC50 value was higher than 1000 mg/kg dry soil weight.

Executive summary:

Expressed as the nominal concentration of the test substance, the lethal effect of SENSANOV WR (SPQP20101202-0165) on Eisenia foetida is as following: The 7 days LC50 value was higher than 1000 mg/kg dry soil weight. The 14 days LC50 value was higher than 1000 mg/kg dry soil weight.

Description of key information

Two test of toxicity to soil macroorganisms have been performed on the registered substance: an OECD Guideline 207 Earthworm, acute toxicity test and an OECD Guideline 222 Earthworm Reproduction Test.

The acute toxicity test (OECD 207) has shown no effects at the highest concentration (LC50 (14d) > 1000 mg/kg dw soil). And in the chronic toxicity test (OECD 222), no effects were observed at the highest tested concentration (1000 mg/kg dw soil):

The LC50 could not be established due to a lack of effect and was therefore estimated to be >1000 mg a.i./kg dry soil. The NOEC for 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 established due to a lack of adverse effect. The EC50 was therefore estimated 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 established due to a lack of a adverse effect. The EC50 was therefore estimated to be >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

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

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

1 000 mg/kg soil dw