2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2-methylphenyl)-3-oxobutyramide]

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:

2009-11-19 to 2010-01-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

DIN ISO 11268-2: 1998 (E), BBA Guideline (1994)

Deviations:

yes

Remarks:

Room temperature decreased several times to a minimum of 17 °C for a total time of about 6 hours. The soil moisture of all test vessels deviated by more than 10 % of the initial moisture at the end of the test.

GLP compliance:

yes (incl. QA statement)

Remarks:

According to German Chemikaliengesetz and Directive 88/320/EEC

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The respective amount of the test item was weighed out for the limit concentration, dissolved in acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). The mixture of sand and test item and 778 g demineralised water were added to the artificial soil to achieve a moisture of 54 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and 600 g soil dry weight were filled into each test vessel.
- Controls: Artificial soil with addition of quartz sand moistened with demineralised water without test item was used as control medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): organic solvent, acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL per replicate
- Evaporation of vehicle before use: Yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.47 g


ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

56 d

Remarks:

Removement of adults after 28 days

Test temperature:

20 +/- 2°C

pH:

Application rate
[mg test item/kg soil dry weight] pH-value
day 0 day 56
Control 6.37 6.88
Solvent Control 6.44 6.94
1000 6.30 6.46

Moisture:

Application rate
[mg test item/kg soil dry weight] Moisture [%] of dry weight

day 0 day 56
Control 28.3 34.8*
Solvent Control 27.5 35.1*
1000 28.4 33.7*

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm3, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g DW
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 8
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.35 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 54
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 8 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 523 +/- 61 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 56 days

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration

Nominal and measured concentrations:

Nominal application rate: 1000 mg test substance/kg soil dry weight (DW)

Reference substance (positive control):

yes

Remarks:

1.0 - 2.5 - 5.0 mg Carbendazim/ kg soil DW

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

, mortality, body weight

Details on results:

- Mortality at end of exposure period: No noteworthy mortality of adult earthworms was observed in the control as well as at the test item concentration of 1000 mg test item/kg soil dry weight after 28 days of exposure
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: At test start the earthworms had an individual weight of 0.30 - 0.60 g. The body weights of the earthworms increased in both controls and at the limit test item concentration of 1000 mg test item/kg soil dry weight throughout the first 28 days. The biomass did not differ statistically significant compared to the pooled control at the limit test item concentration.
- No. of offspring produced: The reproduction rate was 297 in the pooled control and 343 at the limit test item concentration of 1000 mg/kg soil dry weight. Compared to the control the reproduction of the earthworms was not statistically significantly reduced at the test item concentration of 1000 mg test item/kg soil dry weight. However, there was a statistically significant increase in earthworm reproduction at the limit test item concentration compared to the pooled control.
The coefficient of variation calculated for the reproduction of both controls was lower than
30 %.
- Behavioural abnormalities: No evident symptoms or changes in the behaviour of the earthworms were observed in the controls as well as at the limit test item concentration of 1000 mg test item/kg soil dry weight after 28 days of exposure
- Other biological observations:None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 2.5 mg Carbendazim/kg soil DW

Reported statistics and error estimates:

t-tests were carried out for the determination of statistically significant differences compared to the control. A Normality Test and an Equal Variance Test were conducted prior to running the t- tests. P-values for both Normality and Equal Variance Test were 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) was a = 0.05.

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate [mg test item/kg soil dry weight]	Mortality [%]
	Replicate
	1	2	3	4	5	6	7	8	Mean
Control	0	0	0	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0	0	10	1.25
1000	0	0	0	0	0	0	0	0	0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate [mg test item/kg soil dry weight]	Effect	Replicate
		1	2	3	4	5	6	7	8
Control	A)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
Solvent Control	A)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	9/10
	H)								1/10
1000	A)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	9/10
	D)								1/10

A): no obersvaton of symptoms or behavioural changes; H) dead earthworm; D) spontaneous segmentation and separation

Body Weight Changes of the Adult Earthworms      

Application rate [mg test item/ kg soil dry weight]	Rep.	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sign.*
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.474	0.699	0.23	47.5	0.23 ± 0.04	47.8 ± 7.81	-
	2	0.476	0.694	0.22	45.8
	3	0.485	0.721	0.24	48.7
	4	0.493	0.726	0.23	47.3
	5	0.483	0.635	0.15	31.5
	6	0.456	0.683	0.23	49.8
	7	0.456	0.701	0.25	53.7
	8	0.473	0.750	0.28	58.6
Solvent Control	1	0.492	0.747	0.26	51.8	0.25 ± 0.04	53.0 ± 8.34	-
	2	0.474	0.667	0.19	40.7
	3	0.485	0.712	0.23	46.8
	4	0.476	0.725	0.25	52.3
	5	0.480	0.705	0.23	46.9
	6	0.458	0.737	0.28	60.9
	7	0.468	0.741	0.27	58.3
	8	0.460	0.763	0.30	65.9
1000	1	0.455	0.722	0.27	58.7	0.25 ± 0.04	52.3 ± 8.77	No
	2	0.491	0.761	0.27	55.0
	3	0.468	0.736	0.27	57.3
	4	0.473	0.751	0.28	58.8
	5	0.483	0.688	0.21	42.4
	6	0.486	0.700	0.21	44.0
	7	0.483	0.675	0.19	39.8
	8	0.457	0.742	0.29	62.4

Rep:= Replicate; Sign. = Significance

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg test item/kg soil dry weight]	Rep.	Number of juveniles	Mean ± SD	CV               [%]	[%] of pooled control	Significance compared to pooled control
Control	1	284	304 ± 41.9	13.8	-	-
	2	300
	3	382
	4	257
	5	270
	6	350
	7	287
	8	298
Solvent Control	1	274	291 ± 44.9	15.4	-	-
	2	251
	3	330
	4	307
	5	333
	6	321
	7	205
	8	310
1000	1	352	343 ± 23.8	6.93	115	Yes
	2	293
	3	340
	4	377
	5	339
	6	342
	7	357
	8	344

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test item did not induce evident mortality or pathological symptoms of adult earthworms after an exposure to the test item in a concentration of 1000 mg/kg soil dry weight for 28 days. Also, the biomass increase of the adult earthworms after 28 days was not statistically significantly reduced compared to the pooled control at the test item concentration of 1000 mg test item/kg soil dry weight. After eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at the limit test item concentration of 1000 mg test item/ kg soil dry weight compared to the pooled control.

Thus, the No Observed Effect Concentration (NOEC) with respect to mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be at 1000 mg test item/kg soil dry weight.

Executive summary:

Effects of the test item on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) from November 19^th, 2009 to January 16^th, 2010 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany. The study was conducted under static conditions over 8 weeks with a limit test item concentration of 1000 mg test item/kg soil dry weight, which was mixed into artificial soil containing 10 % peat. A control using untreated artificial soil and a solvent control with the solvent mixed into the artificial soil were set up. 80 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 8 replicate groups with 10 test organisms per replicate.

After 28 days no evident mortality as well as no pathological symptoms or changes in the behaviour of adult earthworms were seen in the controls and at the limit test item concentration of 1000 mg test item/kg soil dry weight. The earthworm biomass increased in the controls and at the limit test item concentration of 1000 mg test item/kg soil dry weight over 28 days without any statistically significant differences between pooled control and limit test concentration.

After further four weeks the reproduction rate (average number of juveniles) was 297 in the pooled control and 343 at the limit test item concentration. Compared to the pooled control, the reproduction rate was not statistically significantly reduced, but increased at 1000 mg test item/kg soil dry weight.

Overall, the NOEC of the test item concerning earthworm mortality, biomass and reduction of reproduction was determined to be at 1000 mg test item/kg soil dry weight.

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Tested Dose 

Effects	Test item [mg test item/kg soil dry weight]
Mortality of adult earthworms	> 1000
Reduction of body weight	> 1000
Reduction of reproduction rate   (Number of juveniles)	> 1000
Summary of all effects	> 1000

NOEC and LOEC for Reproduction, Mortality and Body Weight

Test item
	Application rate
	[mg test item/kg soil dry weight]
NOECreproduction, mortality, body weight	1000
LOECreproduction, mortality, body weight	> 1000

Description of key information

Under the conditions of this study, the test item did not induce evident mortality or pathological symptoms of adult earthworms after an exposure to the test item in a concentration of 1000 mg/kg soil dry weight for 28 days. Also, the biomass increase of the adult earthworms after 28 days was not statistically significantly reduced compared to the pooled control at the test item concentration of 1000 mg test item/kg soil dry weight. After eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at the limit test item concentration of 1000 mg test item/ kg soil dry weight compared to the pooled control.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw

Additional information

The results from the two long term toxicity tests on Eisenia fetida with Pigments Yellow 12 and its structural analogue, Pigment Yellow 83, conclusively show absence of toxicity to earth worms, neither in regard to reproduction, nor in regard to mortality and body weight.

As long term toxicity tests towards soil macro-organisms except arthropods (earth worm Eisenia fetida) for two different pigments of the Diarylide Yellow group had been conducted with test substance concentrations far above the solubility limit in water, from acute tests no additional information could be expected. Therefore, no acute toxicity tests were performed.

Judging from these long-term toxicity tests with two different pigments, the Diarylide Yellow Pigments are to be regarded as nontoxic to soil macroorganisms.