2-[(2-methoxy-4-nitrophenyl)azo]-N-(2-methoxyphenyl)-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

Reliability:

1 (reliable without restriction)

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:

The room temperature decreased several times to a minimum of 17 °C for a total time of about 17 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

Analytical monitoring:

no

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The respective test item amount for the limit test item concentration was weighed out, dispersed in acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent the treated quartz sand was given to the artificial soil. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 56 % of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and 600 g soil dry weight was filled into each test vessel.
- Controls: Artificial soil with addition of quartz sand treated with acetone and moistened with demineralised water without test item was used as solvent 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): 0.50 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:

17 - 22 °C

pH:

Application rate
[mg test item/kg soil dry weight] pH-value
day 0 day 56
Control 5.96 5.91
Solvent Control 5.89 5.89
1000 5.81 5.82

Moisture:

Application rate
[mg test item/kg soil dry weight] Moisture [%] of dry weight WHCmax of artificial soil
[g/100 g DW]
day 0 day 56
Control 35.4 39.9* 66
Solvent Control 33.8 42.6*
1000 32.9 40.2*

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, 0.4 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 66
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 4 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 490 +/- 56.8 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

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

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:

reduction of body weight, mortality of adult earthworms

Details on results:

- Mortality at end of exposure period: No noteworthy mortality of adult earthworms was observed in the controls 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 biomass of the adult earthworms increased in the controls and at the limit test item concentration of 1000 mg test item/kg soil dry weight over 28 days of exposure with a statistically significant higher increase at 1000 mg test item/kg soil dry weight compared to the pooled control
- No. of offspring produced: The reproduction rate was 395 in the pooled control and 384 at the limit test item concentration of 1000 mg/kg soil dry weight. Compared to the pooled control the reproduction of the earthworms did not statistically significantly differ at the test item concentration of 1000 mg test item/kg soil dry weight
- Behavioural abnormalities: No 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 pooled 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	10	0	0	0	0	0	0	0	1.25
Solvent Control	0	0	0	0	0	10	0	0	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)	9/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10
	H)	1/10	–	–	–	–	–	–	–
Solvent Control	A)	10/10	10/10	10/10	10/10	10/10	9/10	10/10	10/10
	H)	–	–	–	–	–	1/10	–	–
1000	A)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10

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	0.533	0.768	0.23	44.0	0.22 ± 0.02	43.5 ± 5.22	–
	2	0.501	0.700	0.20	39.7
	3	0.513	0.700	0.19	36.5
	4	0.487	0.699	0.21	43.5
	5	0.500	0.694	0.19	38.8
	6	0.502	0.765	0.26	52.4
	7	0.481	0.694	0.21	44.3
	8	0.470	0.698	0.23	48.5
Solvent Control	1	0.495	0.667	0.17	34.7	0.20 ± 0.04	41.4 ± 8.72	–
	2	0.527	0.694	0.17	31.7
	3	0.491	0.687	0.20	39.9
	4	0.508	0.737	0.23	45.1
	5	0.501	0.674	0.17	34.5
	6	0.484	0.747	0.26	54.3
	7	0.470	0.722	0.25	53.6
	8	0.499	0.686	0.19	37.5
1000	1	0.510	0.728	0.22	42.7	0.24 ± 0.01	48.7 ± 2.73	Yes*
	2	0.484	0.736	0.25	52.1
	3	0.528	0.786	0.26	48.9
	4	0.502	0.741	0.24	47.6
	5	0.498	0.745	0.25	49.6
	6	0.503	0.754	0.25	49.9
	7	0.484	0.722	0.24	49.2
	8	0.472	0.708	0.24	50.0

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg test item/kg soil dry weigt]	Rep.	Number of juveniles	Mean ± SD	CV               [%]	[%] of pooled control	Significance compared to pooled control
Control	1	268	384 ± 54.3	14.1	–	–
	2	429
	3	394
	4	394
	5	347
	6	400
	7	401
	8	439
Solvent Control	1	497	406 ± 58.2	14.3	–	–
	2	453
	3	421
	4	317
	5	430
	6	365
	7	353
	8	411
1000	1	478	384 ± 50.9	13.3	97.2	No
	2	429
	3	355
	4	322
	5	409
	6	349
	7	367
	8	363

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 limit item concentration of 1000 mg test item/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 limit test item concentration of 1000 mg test item/kg soil dry weight. After eight weeks of exposure the earthworm reproduction did not statistically significantly differ to the control at the limit test item concentration of 1000 mg test item/kg soil dry weight.

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 per kg soil dry weight.

Executive summary:

Effects of Pigment Yellow 74on mortality, biomass and the reproductive potential of the earthworm speciesEisenia fetida(Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994)The study was conducted under static conditions over 8 weeks with the limit test item concentration of 1000 mg test item/kg soil dry weight.

The NOEC of the test item concerning mortality, biomass and reproduction of earthworms was determined to be at1000 mg test item/kg soil dry weight.

Summary of All Observed Effects in the Tested Dose 

Effects	[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

Effects of Pigment Yellow 74on mortality, biomass and the reproductive potential of the earthworm speciesEisenia fetida(Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994)The study was conducted under static conditions over 8 weeks with the limit test item concentration of 1000 mg test item/kg soil dry weight.

The NOEC of the test item concerning mortality, biomass and reproduction of earthworms was determined to be at1000 mg test item/kg soil dry weight.