Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

November 1991 to January 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU & OECD test guidance in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.8 (Toxicity for Earthworms: Artificial Soil Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

EC Number:

413-550-5

EC Name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

Cas Number:

142068-96-0

Molecular formula:

Hill formula: C53H28Cl2N14Na8O26S8

IUPAC Name:

octasodium 5-[(4-chloro-6-{[4-({[4-chloro-6-({7-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-8-hydroxy-3,6-disulfonatonaphthalen-1-yl}amino)-1,3,5-triazin-2-yl]amino}methyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-3-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Details on test material:

H112339 :- The test chemical (reference: NBY 405/74) was obtained from ICI Central Notification Unit, Alderley Park, Macclesfield, Cheshire SK10 4TJ. A Certificate of Analysis (reference 9108127, dated 8 August 1991) stated that the test sample contained 10.5Z w/w water, therefore dose calculations treated the material as 89.5Z w/w technical material. Macroecology ref. 91/78.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
None

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

The solutions were applied to 1.25 kg and 2.5 kg (dry weight equivalents) of artificial soil for the initial and definitive tests, respectively. Soil concentrations for H112339 in the initial test were nominally 0, 1, 10, 100 and 1,000 mg active ingredient (ai) kg-1 artificial soil (dry weight). In the definitive test the soil concentrations were 0, 100 and 1,000 mg ai kg-1 .

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

The test soil was a mixture of the following ingredients in oven (approximately 105°C) dry weight proportions:
70% fine silica sand (Grade 110) Supplied by British Industrial Sands, Redhill, Surrey, U.K.
20% kaolinite clay (GTY Powder) Supplied by English China Clay International, London S.W.I.
10% peat (Shamrock Moss) organic matter = 97.7% Supplied by Bord na Mona, Dublin, Eire. Ground then 2mm sieved to remove large plant remains.
Calcium carbonate was incorporated into the soil at 5 g kg-1. Ingredients, in their natural fresh weight form, were mixed in a rotary cement mixer.

The application and incorporation of the H112339 test solution in the artificial soil followed the same procedure for both the tests. The weighed artificial soil was placed into a food mixer bowl and the test solution was added slowly while mixing. The soil was mixed for two minutes by a heavy duty food mixer to evenly incorporate the test substance into the soil. Deionised water was then added to bring the moisture content of the soil up to 50% and mixing continued for a further two minutes. 750 g (fresh weight) aliquots were weighed out into 1 litre glass beakers. For the initial and definitive tests, E. foetida were exposed in two and four aliquots (replicates) of treated soil per treatment, respectively.

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Eisenia foetidaused were clitellate adults that had been cultured in bulb fibre compost (J. Arthur Bower's .Lincoln .England) at Jealott's Hill. The earthworms were supplied by Monkfield, The Aquatic Centre, Cambridge, U.K. The cultures arrived at Jealott's Hill between March and May 1990 and were subsequently held in a controlled environment room.

The mean weight + standard deviation (SD) for E. foetida used in the initial and definitive test was 0.55 + 0.03 g and 0.47 + 0.02 g, respectively.

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

No post exposure observation period

Test conditions

Test temperature:

20 + 2°C

pH:

6.1 + 0.3

Moisture:

45-52%

Details on test conditions:

Preparation of Test Solutions.
H112339 solutions were prepared in deionised water, and made up to 25 ml by the addition of deionised water, for application to the artificial soil. 25 ml of deionised water was used in the preparation of the control soil.
The solutions were applied to 1.25 kg and 2.5 kg (dry weight equivalents) of artificial soil for the initial and definitive tests, respectively. Soil concentrations for H112339 in the initial test were nominally 0, 1, 10, 100 and 1,000 mg active ingredient (ai) kg-1 artificial soil (dry weight). In the definitive test the soil concentrations were 0, 100 and 1,000 mg ai kg-1 .

Chloroacetamide solutions were prepared in deionised water and made up to 50ml for application to artificial soil, to give nominal concentrations of 18, 32 and 56 mg kg-1 (dry weight) as a toxic standard treatment. Chloroacetamide was tested on this batch of E. foetida on 16 August 1991.

Application and Incorporation of Test Solutions in Artificial Soil
The application and incorporation of the H112339 test solution in the artificial soil followed the same procedure for both the tests. The weighed artificial soil was placed into a food mixer bowl and the test solution was added slowly while mixing. The soil was mixed for two minutes by a heavy duty food mixer to evenly incorporate the test substance into the soil. Deionised water was then added to bring the moisture content of the soil up to 50% and mixing continued for a further two minutes. 750 g (fresh weight) aliquots were weighed out into 1 litre glass beakerB. For the initial and definitive tests, E. foetida were exposed in two and four aliquots (replicates) of treated soil per treatment, respectively.

Addition of Test Earthworms
Clitellate E. foetida, which had been preconditioned in untreated artificial soil (50% moisture content) for the previous 24 hours, were added, ten to each beaker. Each batch of ten E. foetida was weighed so that any weight change at the end of the exposure period could be measured. The mean weight and standard deviation of E. foetida used in the test was determined from these weighings. The E. foetida were placed on the surface of the soil between 1 and 2 hours after chemical treatment, and cling film was placed over the top to prevent them from escaping. Six small holes were made in the cling film to allow ventilation.

Test Conditions
The conditions throughout the tests were as follows:

Soil moisture contents were within the range 45-52% throughout the test period (measured at the beginning and end of the tests). Moisture contents were maintained by surface watering with deionised water. The pH of the soils remained within the range 6.1 + 0.3. E. foetida in the test soil were held in a constant environment room at a temperature of 20 + 2°C. A 24 hour light regime was maintained from six fluorescent white tubes. Light intensity was 1,000 + 300 lux.
Assessment of Earthworms

E. foetida were assessed for mortality and any abnormal behavioural effects after 14 days in the initial test and after 7 and 14 days in the definitive test. The soil was tipped onto a clean surface and E. foetida were recorded as dead if there was no response to a mechanical stimulus of their front end. Any E. foetida not found after careful searching of the soil were assumed to be dead. Each batch of ten E. foetida was weighed at the beginning and end of each test to determine any change in bodyweight.

Nominal and measured concentrations:

Initial test were nominally 0, 1, 10, 100 and 1,000 mg active ingredient (ai) kg-1 artificial soil (dry weight). In the definitive test the soil concentrations were 0, 100 and 1,000 mg ai kg-1.

Reference substance (positive control):

yes

Remarks:

2-chloroacetamide

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 other: mg ai kg-1

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: mg ai kg-1

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

The 14 day LC50 value for H112339 was greater than 1,000 mg ai kg-1 (the highest dose tested). There were no mortalities, behavioural effects nor significant bodyweight changes compared to the control (P=5%) at 1,000 mg ai kg-1 in the definitive test. However, all earthworms in this treatment showed red coloration. This was not considered to be a toxic effect. At 100 mg ai kg-1 there were no deaths, abnormalities in behaviour or external condition, nor were there any significant effects on bodyweight (P=5%). Hence, the No Observed Effect Level was at least 100 mg ai kg-1. There was no control mortality.

Results with reference substance (positive control):

The 14 day LC50 value (and 95% confidence limits) for 2-chloroacetamide was 38 (35-43) mg kg-1.

Reported statistics and error estimates:

The LC50 value and its 95% confidence limits were estimated for chloroacetamide using the standard technique of iteratively reweighted linear regression of the logit of percent mortality upon log10 (dose).

For H112339, the data on bodyweight were analysed by a one-way analysis of covariance using final (post-treatment) bodyweight as the dependent variable and initial (pre-treatment) bodyweight as a covariate. In this analysis it was assumed that there was a straight line relationship between final and initial bodyweights. The effect of this analysis is to adjust the means of the final bodyweights to a constant value of initial weight so that a meaningful comparison can be made between them.

Using the pooled estimate of error variability from the analysis, t-tests were carried out between the mean final bodyweight (adjusted for the mean initial bodyweight) at each dose with the mean final bodyweight (adjusted for the mean initial bodyweight) in the control.

LC50 estimation was performed using the in-house package LOGITPC Version 1.2. Analysis of covariance was performed using Statistical Analysis System (SAS), Version 6.04.

Any other information on results incl. tables

Initial Test – Mortality and bodyweight ofE. foetidaafter 14 days exposure to H112339 in artificial soil

H112339 Concentration in Soil (mg ai kg^-1)	Replicate	Numbers dead out of 10	Weight of 10E. foetida(g)
		14 days	Start Day 0	Finish Day 14	% Change
Control	1	0	5.8	5.6	-3.4
	2	0	5.8	5.4	-6.9
1 mg kg^-1	1	0	5.6	5.2	-7.1
	2	0	5.7	5.0	-12.3
10 mg kg^-1	1	0	5.0	4.8	-4.0
	2	0	5.2	4.8	-7.7
100 mg kg^-1	1	0	5.4	5.1	-5.6
	2	0	5.9	5.3	-10.2
1000 mg kg^-1	1	1	5.6	5.2^a	-7.1
	2	1	5.2	5.0^a	.3.8

a            Where mortality occurred, the end weight and the percentage weight were adjusted to account for the dead earthworm using the following calculation:

               End weight for 10 worms (g) =

               Weight of remaining worms (g) X No. Exposed (10)/No. Surviving

 

Definitive Test – Mortality and bodyweight ofE. foetidaafter 7 and 14 days exposure to H112339 in artificial soil

H112339 Concentration in Soil (mg ai kg^-1)	Replicate	Numbers dead out of 10		Weight of 10E. foetida(g)
		7 days	14 days	Start Day 0	Finish Day 14	% Change
Control	1	0	0	4.4	4.3	-2.3
	2	0	0	5.0	4.7	-6.0
	3	0	0	4.4	4.4	0
	4	0	0	4.4	4.4	0
100 mg kg^-1	1	0	0	4.7	4.5	-4.3
	2	0	0	4.5	4.5	0
	3	0	0	5.1	5.2	+2.0
	4	0	0	4.9	4.9	0
1000 mg kg^-1	1	0	0	4.7	4.4	-6.4
	2	0	0	4.5	4.2	-6.7
	3	0	0	4.9	4.6	-6.1
	4	0	0	4.6	4.7	+2.2

 

Mortality ofE. foetidaafter 7 and 14 days exposure to 2-chloroacetamide in artificial soil

Chloroacetamide Concentration in Soil (mg kg^-1)	Replicate	Numbers Dead out of 10
		7 days	14 days
18	1	0	0
	2	0	0
	3	0	0
	4	0	0
32	1	2	2
	2	0	0
	3	1	3
	4	1	1
56	1	10	10
	2	10	10
	3	10	10
	4	9	9

 

Treatment Means ofE. foetidabodyweights after 14 days exposure to H112339

Treatment	Mean Adjusted Final Bodyweights and (Standard Errors) for Groups of 10 Earthworms
Control	4.55 (0.09)
100 mg ai kg^-1	4.67 (0.09)
1000 mg ai kg^-1	4.48 (0.08)

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 14 day LC50 value for H112339 was greater than 1,000 mg ai kg-1 which was the highest dose tested. The No Observed Effect Level was at least 100 mg ai kg-1.

Executive summary:

Eisenia foetida(clitellate adults) were exposed to concentrations of test substance BT12339 evenly incorporated into an artificial soil for 14 days at 20 + 2°C.

 The 14 day LC50 value was greater than 1,000 mg active ingredient (ai) kg^-1soil which was the highest dose tested.   At 100 mg ai kg^-1there were no deaths or significant bodyweight changes (P=5%) nor abnormalities in behaviour or external condition.   Thus, the No Observed Effect Level was at least 100 mg ai kg^-1.   The only effect at 1,000 mg ai kg^-1in the definitive test was the red coloration of the earthworms which was not considered to be a toxic effect.

 

The test substance is not considered to pose a hazard to soil macro-organisms.