4-[(2,5-dichlorophenyl)azo]-3-hydroxy-N-phenylnaphthalene-2-carboxamide

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:

2012-03-15 to 2012-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to relevant guidelines and compliant to GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Food was provided on day 0 instead of day 1. This deviation is considered to have no impact on quality and integrity of the study. During 1 day the room temperature increased to approx 23°C. During 1 day the room temperature decreased below 18°C. These d

Principles of method if other than guideline:

Food was provided on day 0 instead of day 1. This deviation is considered to have no impact on quality and integrity of the study.
During 1 day the room temperature increased to approx 23°C. During 1 day the room temperature decreased below 18°C. These deviations are considered to have no impact on the quality and integrity of the study.
Variation of the soil water content slightly > 10%: This deviation is considered to have no impact on the quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt 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 test item amount was weighed out for the limit test item concentration, dissolved 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. Additional demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 52 % of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed to ensure a homogeneous distribution and about 600 g soil dry weight were filled into each test vessel.
- Controls: Artificial soil moistened with demineralised water without test or reference item and without solvent was used as control medium.
- Solvent control: Artificial soil with addition of quartz sand treated with acetone and moistened with demineralised water without test item was used as solvent control.

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): 0.44 g, 0.3-0.6 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:

Removal of adults after 28 days

Test temperature:

16* - 23*
*) During 1 day the room temperature increased to approx 23°C. During 1 day the room temperature decreased
below 18°C. These deviations are considered to have no impact on the quality and integrity of the study. For the remaining days temperature was at 20 +/- 2 deg C.

pH:

Application rate
[mg / kg soil dry weight] pH-value
day 0 day 56
Control 5.71 6.35
Solvent Control 5.71 6.27
1000 5.53 6.17

Moisture:

Application rate
[mg / kg soil dry weight] Moisture [%] of dry weight
day 0 day 56
Control 33.1 37.4*
Solvent Control 32.9 37.1*
1000 33.2 36.8*

*) Variation of the water content slightly > 10%: This deviation is considered to have no impact on the quality and integrity of the study.

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 (80)
- No. of replicates per treatment group: 8
- No. of replicates per control: 8



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.41 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 65
- 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: 548 +/- 83 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:

, mortality, body weight

Details on results:

- Mortality at end of exposure period:
No mortality of adult earthworms in the controls and at the limit test item concentration was observed after 28 days of exposure to the test item
- 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 0.37 - 0.54 g. As there were no statistically significant differences determined between the control and the solvent control for the body weight, the controls were pooled. The body weights of the adult earthworms increased throughout the first 28 days at the limit application rate with no statistically significant differences compared to the pooled control.
- No. of offspring produced:
As there were no statistically significant differences determined between the control and the solvent control for the reproduction rate, the controls were pooled.
The mean number of juveniles in the pooled control was 185. The average number of juveniles in the treated group was 164. Compared to the pooled control the reproduction of the earthworms did not statistically significantly differ
- Behavioural abnormalities:
No pathological symptoms nor changes in the behaviour of the earthworms were observed in the controls or in the limit test item application rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: Determined EC50 = 1.31 mg Carbendazim/kg soil DW (a.i.), which is within the range recommended by the guideline.

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	0	0	0	0	0	0	0	0	0
Solvent Control	0	0	0	0	0	0	0	0	0
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	10/10
1000	A)	10/10	10/10	10/10	10/10	10/10	10/10	10/10	10/10

A) No observation of symptoms or behavioural changes.

Body Weight Changes of the Adult Earthworms      

Application rates [mg test item /kg soil dry weight]	Replicate	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig. to pooled control
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.43	0.66	0.23	53.5	0.19 ± 0.03	43.5 ± 7.91	-
	2	0.44	0.60	0.16	36.4
	3	0.42	0.65	0.23	54.8
	4	0.45	0.64	0.19	42.2
	5	0.43	0.64	0.21	48.8
	6	0.44	0.59	0.15	34.1
	7	0.42	0.59	0.17	40.5
	8	0.45	0.62	0.17	37.8
Solvent Control	1	0.45	0.63	0.18	40.0	0.17 ± 0.03	39.1 ± 5.98	-
	2	0.43	0.64	0.21	48.8
	3	0.44	0.58	0.14	31.8
	4	0.42	0.58	0.16	38.1
	5	0.45	0.66	0.21	46.7
	6	0.43	0.58	0.15	34.9
	7	0.44	0.59	0.15	34.1
	8	0.42	0.58	0.16	38.1
Pooled Control						0.18 ± 0.03	41.3 ± 7.15	-
1000	1	0.42	0.60	0.18	42.9	0.19 ± 0.03	44.6 ± 7.59	No
	2	0.45	0.64	0.19	42.2
	3	0.43	0.67	0.24	55.8
	4	0.44	0.60	0.16	36.4
	5	0.42	0.66	0.24	57.1
	6	0.45	0.64	0.19	42.2
	7	0.43	0.60	0.17	39.5
	8	0.44	0.62	0.18	40.9

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	149	173 ± 41.9	24.2	–	–
	2	259
	3	131
	4	199
	5	156
	6	151
	7	193
	8	146
Solvent Control	1	177	197 ± 36.8	18.7	–	–
	2	140
	3	168
	4	233
	5	230
	6	172
	7	231
	8	226
Pooled Control			185 ± 40.1	21.7	–	–
1000	1	244	164 ± 51.5	31.4	88.6	No
	2	236
	3	112
	4	144
	5	170
	6	167
	7	129
	8	113

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test item did not induce mortality, pathological symptoms or changes in the behaviour of adult earthworms after 28 days of exposure at the nominal limit test item concentration of 1000 mg/kg soil dw. The adult earthworm biomass increased in the control and at the limit test item concentration without any statistically significant differences. After eight weeks of exposure the earthworm reproduction at the limit test item concentration did not statistically significantly differ from the pooled control. As there had been no statistically significant differences determined between the control and the solvent control for the body weight and the reproduction rate, the controls were pooled.

Thus, the No Observed Effect Concentration (NOEC) for the test item summarizing the endpoints mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be 1000 mg /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 Guideline OECD 222 (2004). 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, that was mixed into artificial soil containing 10 % peat. An untreated control and a solvent control were set up. 80 test organisms were inserted into 8 control, solvent control and test item replicates (10 earthworms per replicate). They had an individual body weight between 0.37 and 0.54 g at experimental starting.

After 28 days of exposure in soil, no earthworm mortalities as well as no pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. As there were no statistically significant differences determined between the control and the solvent control for the body weight and the reproduction rate, the controls were pooled. After 28 days of exposure, no statistically significant differences in earthworm body weights at 1000 mg/kg soil dry weight were observed. After further four weeks the reproduction rate (average number of juveniles produced) was 185 in the pooled control and ranged between 112 and 244 at the limit test item concentration. Compared to the pooled control, reproduction rate was not statistically significantly reduced at 1000 mg/kg soil dry weight.

Overall, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms was 1000 mg/kg soil dry weight.

All validity criteria recommended by the test guideline were fulfilled.

Summary of All Observed Effects in the Tested Application Rate

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

Table 2:                 NOEC and LOEC values

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

PR002

Under the conditions of this study, the test item did not induce mortality, pathological symptoms or changes in the behaviour of adult earthworms after 28 days of exposure at the nominal limit test item concentration of 1000 mg/kg soil dw. The adult earthworm biomass increased in the control and at the limit test item concentration without any statistically significant differences. After eight weeks of exposure the earthworm reproduction at the limit test item concentration did not statistically significantly differ from the pooled control. As there had been no statistically significant differences determined between the control and the solvent control for the body weight and the reproduction rate, the controls were pooled.

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

Key value for chemical safety assessment

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

1 000 mg/kg soil dw