N-(5-chloro-2-methoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-06-10 to 2009-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study compliant to GLP

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 increased once to about 22.5 °C for four hours. The soil moisture of all test vessels deviated by more than 10 % of the initial value at the end of the test.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP 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 test item concentration was weighed out, 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. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 52 % of the maximum water holding capacity. Subsequently, the test medium will be mixed with a mixer for at least three minutes 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.
- 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 acetone per replicate, 10 g quartz sand 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): 0.44 +/- 0.05 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:

no

Total exposure duration:

56 d

Remarks:

Removement of adults after 28 days

Test temperature:

18 - 22.5 deg C

pH:

Application rate
[mg test item/kg soil DW] pH-value
day 0 day 56
Control 5.42 6.13
Solv. Control 5.40 6.40
62.5 5.39 6.18
125 5.39 6.30
250 5.40 6.33
500 5.38 6.07
1000 5.42 6.14

Moisture:

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

day 0 day 56
Control 35.6 42.5*
Solv. Control 35.1 45.8*
62.5 34.8 46.7*
125 35.5 46.8*
250 34.9 44.6*
500 35.2 45.2*
1000 34.6 44.6*

*Variation of the water content > 10 %, this deviation is considered to have no impact on 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 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4


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): 68
- 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: 524 +/- 70 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: 2

Nominal and measured concentrations:

Nominal application rates: 62.5 - 125 - 250 - 500 - 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 mortality of adult earthworms was observed in the controls as well as at all application rates 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 0.30 - 0.60 g. The body weights of the earthworms increased in the control and at the test item concentrations of 62.5, 125, 250, 500 and 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 all test item concentrations
- No. of offspring produced: The mean number of juveniles in the pooled control was 359. The average number of juveniles in the treated groups ranged between 288 and 365. Compared to the pooled control the reproduction of the earthworms did not statistically significantly differ at all test item application rates
- Behavioural abnormalities: No symptoms or changes in the behaviour of the earthworms were observed in the controls as well as at all application rates after 28 days of exposure to the test item

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:

Differences between the control and the solvent control were statistically analysed using t-tests. No statistical differences occurred between the controls concerning biomass and reproduction. Thus, both controls were pooled for further statistical analyses.
One Way Analysis of Variance (ANOVA) was 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 ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is 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	Replicate 2	Replicate 3	Replicate 4
Control	0	0	0	0
Solvent Control	0	0	0	0
62.5	0	0	0	0
125	0	0	0	0
250	0	0	0	0
500	0	0	0	0
1000	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
Control	a)	10/10	10/10	10/10	10/10
Solvent Control	a)	10/10	10/10	10/10	10/10
62.5	a)	10/10	10/10	10/10	10/10
125	a)	10/10	10/10	10/10	10/10
250	a)	10/10	10/10	10/10	10/10
500	a)	10/10	10/10	10/10	10/10
1000	a)	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	0.481	0.541	0.06	12.5	0.10 ± 0.03	22.5 ± 6.42	-
	2	0.448	0.541	0.09	20.8
	3	0.439	0.565	0.13	28.7
	4	0.453	0.552	0.10	21.9
Solvent Control	1	0.441	0.570	0.13	29.3
	2	0.479	0.563	0.08	17.5
	3	0.447	0.583	0.14	30.4
	4	0.449	0.534	0.09	18.9
62.5	1	0.479	0.593	0.11	23.8	0.13 ± 0.03	28.2 ± 7.74	No
	2	0.444	0.600	0.16	35.1
	3	0.451	0.605	0.15	34.2
	4	0.452	0.540	0.09	19.5
125	1	0.451	0.580	0.13	28.6	0.13 ± 0.01	28.2 ± 3.63	No
	2	0.451	0.565	0.11	25.3
	3	0.478	0.601	0.12	25.7
	4	0.437	0.582	0.15	33.2
250	1	0.477	0.575	0.10	20.5	0.12 ± 0.03	27.1 ± 6.57	No
	2	0.448	0.594	0.15	32.6
	3	0.453	0.554	0.10	22.3
	4	0.444	0.590	0.15	32.9
500	1	0.444	0.581	0.14	30.9	0.12 ± 0.03	27.3 ± 7.81	No
	2	0.442	0.581	0.14	31.4
	3	0.454	0.596	0.14	31.3
	4	0.475	0.549	0.07	15.6
1000	1	0.454	0.599	0.15	31.9	0.14 ± 0.02	29.9 ± 5.07	No
	2	0.445	0.606	0.16	36.2
	3	0.480	0.606	0.13	26.3
	4	0.441	0.553	0.11	25.4

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	359	359 ± 49.6	13.8	-	-
	2	338
	3	456
	4	323
Solvent Control	1	354
	2	388
	3	287
	4	365
62.5	1	338	365 ± 28.5	7.81	102	No
	2	356
	3	360
	4	405
125	1	284	355 ± 60.2	17.0	98.9	No
	2	327
	3	393
	4	415
250	1	378	347 ± 88.3	25.5	96.6	No
	2	442
	3	334
	4	232
500	1	303	288 ± 49.1	17.1	80.3	No
	2	250
	3	351
	4	248
1000	1	344	346 ± 83.8	24.2	96.4	No
	2	385
	3	424
	4	230

Validity criteria fulfilled:

yes

Conclusions:

In this earthworm reproduction test according to OECD 222 the submission substance did not induce mortality or pathological symptoms of adult earthworms after an exposure to the test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight for 28 days. Also, the biomass increase of the adult earthworms after 28 days did not statistically significantly differ compared to the pooled control at all test item concentrations. In addition, after eight weeks of exposure the earthworm reproduction was not statistically significantly reduced at all applied test item concentrations.

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

Executive summary:

Effects of the submission substance on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined compliant to GLP according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) (reliability category 1). The study was conducted under static conditions over 8 weeks with five different application rates (62.5 - 125 - 250 - 500 - 1000 mg test item/kg soil dry weight), which were mixed into artificial soil containing 10 % peat. 40 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 4 replicate groups with 10 test organisms per replicate.

After 28 days no mortality as well as no pathological symptoms or changes in the behaviour of adult earthworms were seen in the control, in the solvent control and at all tested application rates. The earthworm biomass increased in the control and at all application rates over 28 days without any statistically significant differences compared to the control. After further four weeks the reproduction rate (average number of juveniles) was 359 in the control (control and solvent control) and ranged between 288 and 365 at the test item application rates. Compared to the control, the reproduction rate did not statistically significantly differ at all application rates.

Overall, the NOEC of the submission substance concerning mortality, biomass and reproduction of earthworms was determined to be >= 1000 mg test item/kg soil dry weight (Table 2).

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Tested Doses

Effects	Test item
mg test item/kg soil dry weight	62.5	125	250	500	1000
Mortality of adult earthworms	No	No	No	No	No
Reduction of body weight	No	No	No	No	No
Reduced reproduction rate   (Number of juveniles)	No	No	No	No	No
Summary of all effects	No	No	No	No	No

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

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-21 to 2009-01-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-study compliant to GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

DIN ISO 11268-2 and BBA Guideline

Deviations:

yes

Remarks:

The room temperature decreased three times to about 17.5 °C and increased three times to about 21 °C . The soil moisture of all test vessels deviated by more than 10 %. Deviations considered to have no impact on study quality and integrity.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt and Directive 88/320/EEC

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The respective test item concentration was weighed out, 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. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 52 % of the maximum water holding capacity.
Subsequently, the test medium was mixed with a long handled blender for at least three minutes and filled into the test vessels.
- 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): Quartz sand treated with 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): 0.54 +/- 0.04 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:

no

Total exposure duration:

56 d

Post exposure observation period:

No

Test temperature:

Room Temperature [°C]
nominal 20 +/- 2
actual 17.5 - 21.5*
*) The room temperature decreased three times to about 17.5 deg C for a total of 9 hours and increased three times to about 21 deg C for a total of 12 hours.

pH:

Application rate
[mg test item/kg soil DW] pH-value
day 0 day 56
Control 6.07 6.42
Solv. Control 5.99 6.32
62.5 5.71 6.51
125 5.71 6.65
250 5.71 6.58
500 5.82 6.55
1000 5.69 6.73

Moisture:

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

day 0 day 56
Control 33.5 38.3*
Solv. Control 32.3 39.4*
62.5 32.5 39.9*
125 32.5 39.2*
250 32.4 39.3*
500 32.4 37.2*
1000 31.2 38.9*

*) Variation of the water content > 10 %, this deviation is considered to have no impact on 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 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.36 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 65
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 9 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 614 ± 67.7 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: 2

Nominal and measured concentrations:

nominal concentrations: 62.5 - 125 - 250 - 500 - 1000 mg test item/kg soil DW

Reference substance (positive control):

yes

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 and reduction of body weight

Details on results:

- Mortality at end of exposure period: No mortality was observed in any of the tested test item and control groups
- Total mass of adults at beginning of test: 0.49 - 0.50
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: Control and solvent control: 46.8 +/- 7.47, 62.5 mg/kg soil DW: 46.0 +/- 2.09, 125 mg/kg soil DW: 50.7 +/- 11.3, 250 mg/kg soil DW: 57.1 +/- 8.37, 500 mg/kg soil DW: 56.4 +/- 4.73, 1000 mg/kg soil DW: 57.9 +/- 2.50
- No. of offspring produced: Control AND SOLVENT CONTROL: 235 +/- 39.1, 62.5 mg/kg soil DW: 244 +/- 30.4, 125 mg/kg soil DW: 238 +/- 61.6, 250 mg/kg soil DW: 269 +/- 42.6, 500 mg/kg soil DW: 237 +/-67.5, 1000 mg/kg soil DW: 267 +/- 31.4
- No. of unhatched cocoons: Not determined
- Morphological abnormalities: None
- Behavioural abnormalities: None
- Other biological observations: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Reference Item (Carben) - Reproduction Rate (Number of Juveniles after 8 weeks)
Application rate
[mg a.i./kg soil dry weight] Rep. Number of juveniles Mean ± SD CV Reduction compared to control [%] Significance*
Control 1 248 272
±
34.8 12.8 - -
2 236
3 305
4 298
1.0 1 224 259
±
47.4 18.3 4.60 No
2 263
3 225
4 325
2.5 1 39 44.8
±
13.8 30.9 83.5 Yes
2 65
3 34
4 41
5.0 1 0 0
±
0.0 - 100 Yes
2 0
3 0
4 0
Rep. = Replicate a.i. = Active ingredient SD = Standard deviation CV = Coefficient of variation

Reported statistics and error estimates:

Differences between the control and the solvent control were statistically analysed using One Way Analysis of Variance (ANOVA). No statistical differences occurred between the controls concerning biomass and reproduction. Thus, both controls were pooled for further statistical analyses.
One Way Analysis of Variance (ANOVA) was 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 ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05. Since the Normality Test failed using reproduction data, the data were log transformed for ANOVA to obtain a passed Normality Test.

The EC50-value for reproduction was not calculated since no reduction of reproduction occurred at all application rates.

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

Application rate [mg test item/kg soil dry weight]	Mortality [%]
	Replicate 1	Replicate 2	Replicate 3	Replicate 4
Control	0	0	0	0
Solvent Control	0	0	0	0
62.5	0	0	0	0
125	0	0	0	0
250	0	0	0	0
500	0	0	0	0
1000	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
Control	a)	10/10	10/10	10/10	10/10
Solvent Control	a)	10/10	10/10	10/10	10/10
62.5	a)	10/10	10/10	10/10	10/10
125	a)	10/10	10/10	10/10	10/10
250	a)	10/10	10/10	10/10	10/10
500	a)	10/10	10/10	10/10	10/10
1000	a)	10/10	10/10	10/10	10/10

a) No observation of symptoms or behavioural changes

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	0.498	0.723	0.23	45.2	0.23 ± 0.04	46.8 ± 7.47	-
	2	0.520	0.786	0.27	51.2
	3	0.498	0.672	0.17	34.9
	4	0.500	0.720	0.22	44.0
Solvent Control	1	0.474	0.768	0.29	62.0
	2	0.471	0.685	0.21	45.4
	3	0.496	0.716	0.22	44.4
	4	0.502	0.740	0.24	47.4
62.5	1	0.498	0.720	0.22	44.6	0.23 ± 0.01	46.0 ± 2.09	No
	2	0.503	0.744	0.24	47.9
	3	0.499	0.718	0.22	43.9
	4	0.503	0.743	0.24	47.7
125	1	0.465	0.668	0.20	43.7	0.25 ± 0.05	50.7 ± 11.3	No
	2	0.473	0.791	0.32	67.2
	3	0.500	0.744	0.24	48.8
	4	0.499	0.715	0.22	43.3
250	1	0.471	0.703	0.23	49.3	0.28 ± 0.04	57.1 ± 8.37	No
	2	0.467	0.789	0.32	69.0
	3	0.499	0.776	0.28	55.5
	4	0.503	0.778	0.28	54.7
500	1	0.466	0.754	0.29	61.8	0.27 ± 0.02	56.4 ± 4.73	No
	2	0.470	0.739	0.27	57.2
	3	0.502	0.784	0.28	56.2
	4	0.501	0.753	0.25	50.3
1000	1	0.469	0.727	0.26	55.0	0.28 ± 0.02	57.9 ± 2.50	No
	2	0.470	0.736	0.27	56.6
	3	0.501	0.804	0.30	60.5
	4	0.504	0.803	0.30	59.3

Rep. = Replicate; SD = Standard deviation; Sign. = Significance; * Significance compared to pooled control

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg test item/kg soil DW]	Rep.	Number of juveniles	Mean ± SD	CV               [%]	[%] of pooled control	Significance compared to pooled control
Control	1	227	235 ± 39.1	16.7	-	-
	2	184
	3	203
	4	275
Solvent Control	1	219
	2	211
	3	260
	4	298
62.5	1	279	244 ± 30.4	12.5	104	No
	2	212
	3	258
	4	226
125	1	284	238 ± 61.6	25.8	102	No
	2	165
	3	294
	4	210
250	1	238	269 ± 42.6	15.8	115	No
	2	231
	3	284
	4	322
500	1	182	237 ± 67.5	28.5	101	No
	2	335
	3	209
	4	221
1000	1	299	267 ± 31.4	11.8	114	No
	2	288
	3	236
	4	244

SD = Standard deviation        CV = Coefficient of variation        Rep. = Replicate      

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the submission substance did not induce mortality or pathological symptoms of adult earthworms after an exposure to the test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight for 28 days. Also, the biomass increase of the adult earthworms after 28 days did not statistically significantly differ compared to the pooled control at all test item concentrations. In addition, after 56 days of exposure the earthworm reproduction was not statistically significantly reduced at all applied test item concentrations.

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

Executive summary:

Effects of the submission substance 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) compliant to GLP (reliability category 1). The study was conducted under static conditions over 8 weeks with five different application rates (62.5, 125, 250, 500, and 1000 mg test item/kg soil dry weight), which were mixed into artificial soil containing 10 % peat. 40 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 4 replicate groups with 10 test organisms per replicate.

After 28 days no mortality as well as no pathological symptoms or changes in the behaviour of adult earthworms were seen in the control, in the solvent control and at all tested application rates (Table 1). The earthworm biomass increased in the control and at all application rates over 28 days without any statistically significant differences compared to the control. After further four weeks the reproduction rate (average number of juveniles) was 235 in the control (control and solvent control) and ranged between 237 and 269 at the test item application rates. Compared to the control, the reproduction rate did not statistically significantly differ at all application rates.

Overall, the NOEC of Pigment Red 187 concerning mortality, biomass and reproduction of earthworms was determined to be >=  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 Doses

Effects	Test Item
mg test item/kg soil dry weight	62.5	125	250	500	1000
Mortality of adult earthworms	No	No	No	No	No
Reduction of body weight	No	No	No	No	No
Reduced reproduction rate   (Number of juveniles)	No	No	No	No	No
Summary of all effects	No	No	No	No	No

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

Reference 3

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:

Deviations are decribed in the field 'Principles of method if other than guideline'.

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

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-08 to 2010-03-09

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:

Room temperature decreased several times to a minimum of 17 °C for a total time of about 11 hours. The soil moisture of all test vessels deviated by more than 10 % of the initial moisture at the end of the test since it was decided to add additional water

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 concentration was weighed out, 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. 1164 mL demineralised water were added to adjust the humidity of the artificial soil to a moisture of 54 % of the maximum water holding capacity. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 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.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone, organic solvent
- 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): 0.53 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 5.75 6.25
Solvent Control 5.80 6.45
1000 5.86 6.29

Moisture:

Application rate
[mg test item/kg soil dry weight] Moisture [%] of dry weight
day 0 day 56
Control 37.9 43.8*
Solvent Control 37.3 44.7*
1000 38.2 42.9*

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



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.39 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 73
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 18 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 649 +/- 79 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

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

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 body weights of the earthworms increased in the control and at the limit test item concentration of 1000 mg test item/kg soil dry weight throughout the first 28 days. Compared to the control, no statistically significant difference in the increase of biomass was found
- No. of offspring produced: The mean reproduction rate was 332 in the pooled control and 375 at the limit test item concentration of 1000 mg test item/kg soil dry weight. Compared to the pooled control, the reproduction rate was not statistically significantly reduced, but increased at 1000 mg test item/kg soil dry weight. The coefficient of variation calculated for the reproduction of both controls was lower than 30 %
- 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 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 -value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) was  = 0.05. Due to a failed Normality Test reproduction data were log-transformed prior to running the ANOVA.

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	10	0	0	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)	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	9/10	10/10	10/10	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 [%]	Significance compared to pooled control  with [%] data
		[g]	[g]	[g]	[%]
Control	1	0.573	0.827	0.25	44.3	54.3 ± 7.43	–
	2	0.548	0.805	0.26	46.9
	3	0.498	0.800	0.30	60.6
	4	0.526	0.830	0.30	57.8
	5	0.535	0.799	0.26	49.3
	6	0.494	0.819	0.33	65.8
	7	0.516	0.781	0.27	51.4
	8	0.520	0.822	0.30	58.1
Solvent Control	1	0.524	0.817	0.29	55.9	53.4  ± 9.35	–
	2	0.555	0.805	0.25	45.0
	3	0.553	0.794	0.24	43.6
	4	0.504	0.869	0.36	72.4
	5	0.529	0.811	0.28	53.3
	6	0.535	0.788	0.25	47.3
	7	0.486	0.773	0.29	59.1
	8	0.519	0.780	0.26	50.3
1000	1	0.518	0.790	0.27	52.5	48.0 ±  5.12	No
	2	0.519	0.763	0.24	47.0
	3	0.565	0.831	0.27	47.1
	4	0.545	0.792	0.25	45.3
	5	0.495	0.715	0.22	44.4
	6	0.525	0.779	0.25	48.4
	7	0.525	0.743	0.22	41.5
	8	0.490	0.774	0.28	58.0

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 control	Significance compared to pooled control
Control	1	340	333 ± 35.7	10.7	–	–
	2	299
	3	356
	4	288
	5	378
	6	290
	7	365
	8	350
Solvent Control	1	328	332 ± 56.3	17.0	–	–
	2	293
	3	290
	4	272
	5	339
	6	455
	7	340
	8	335
1000	1	337	375 ± 25.0	6.68	113	Yes*
	2	354
	3	362
	4	395
	5	361
	6	410
	7	390
	8	392

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, Pigment Red 112 did not induce mortality or pathological symptoms of adult earthworms after an exposure to the test item concentration of 1000 mg test item/kg soil dry weight for 28 days. 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 was not statistically significantly reduced, but increased 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/ kg soil dry weight.

Executive summary:

Effects of Pigment Red 112 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). 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 as well as at the limit test item concentration of 1000 mg test item/kg soil dry weight. The biomass of the adult earthworms increased in the pooled control and at the limit test item concentration without a statistically significant difference. After further four weeks the reproduction rate (average number of juveniles) was 332 in the pooled control and 375 at the limit test item concentration. Compared to the pooled control, the reproduction rate was not statistically significantly reduced.

Overall, the NOEC of the test item concerning mortality, biomass and reproduction of earthworms 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

Reference 5

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Refer to the analogue approach provided in the CSR.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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 on result:

other:

Remarks:

Noack, 2010

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

Remarks on result:

other:

Remarks:

Noack, 2009

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 and reduction of body weight

Remarks on result:

other:

Remarks:

Noack, 2009b

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:

mortaility, body weight

Remarks on result:

other:

Remarks:

Noack, 2012

Description of key information

EC10 / NOEC soil macroorganisms ≥ 1000 mg/kg soil dw

Additional information

No toxicity of Pigments Orange 38, Red 2, Red 112 and Red 187 to the earthworm species Eisenia fetida was found in reliable (category 1) Earthworm Reproduction Tests over a period of 8 weeks (OECD 222, 2004). Either tests with a nominal limit concentration of 1000 mg/kg soil dry weight and 80 test organisms divided into 8 replicate groups were performed (Pigment Red 2 and 112), or five test item concentrations from 62.5 to 1000 mg/kg were tested with 40 test organisms per treatment group divided into 4 replicate groups. None of the four tested pigments did induce mortality or pathological symptoms to adult earthworms and the biomass increase of the adult earthworms was not statistically significantly reduced compared to the control after 28 days of exposure. In none of the four tests any statistically significantly reduced reproduction of earthworms was observed compared to the control. All studies were performed compliant to GLP meeting the validity criteria of the OECD guideline. Therefore, Naphtol-AS Pigments are to be regarded as chronically non-toxic to soil macroorganisms.