Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

Toxicity to soil macroorganisms except arthropods

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

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. Deviations considered to have no impact on quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test material

Test material information

Constituent 1

Reference substance name:

Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

EC Number:

256-050-0

EC Name:

Calcium bis[4-[[3-[[2-hydroxy-3-[[(4-methoxyphenyl)amino]carbonyl]-1-naphthyl]azo]-4-methylbenzoyl]amino]benzenesulphonate]

Cas Number:

43035-18-3

Molecular formula:

C32H26N4O7S.1/2Ca

IUPAC Name:

calcium bis(4-{[3-({2-hydroxy-3-[(4-methoxyphenyl)carbamoyl]-1-naphthyl}diazenyl)-4-methylbenzoyl]amino}benzenesulfonate)

Test material form:

solid: nanoform, no surface treatment

Sampling and analysis

Analytical monitoring:

no

Test substrate

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 54 % 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 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

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 design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Removement of adults after 28 days

Test conditions

Test temperature:

18 - 22.5 °C

pH:

Application rate
[mg test item/kg soil dry weight] pH-value
day 0 day 56
Control 5.71 6.28
Solv. Control 5.78 6.31
62.5 5.79 6.27
125 5.69 6.44
250 5.65 6.34
500 5.78 6.22
1000 5.67 6.15

Moisture:

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

day 0 day 56
Control 35.0 44.0*
Solv. Control 35.8 44.6*
62.5 35.3 43.9*
125 36.4 44.0*
250 35.2 44.6*
500 35.2 44.5*
1000 36.2 43.0*
*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: 657 +/- 105 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

Results and discussion

Effect concentrations

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: One dead adult earthworm was found at the application rate of 62.5 mg/kg soil dry weight and two dead earthworms were found at 500 mg/kg soil dry weight after 28 days of exposure. This level of earthworm mortality (2.5 % at 62.5 and 5 % at 500 mg/kg soil dry weight) is not considered to be of biological significance.
- 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 controls and at all test item concentrations throughout the first 28 days without any statistically significant differences compared to the pooled control
- No. of offspring produced: The mean number of juveniles in the pooled control was 367. The average number of juveniles in the treated groups ranged between 349 and 385. Compared to the pooled control the reproduction of the earthworms did not statistically significantly differ at all test item application rates
- Behavioural abnormalities: No pathological symptoms or changes in the behaviour of the adult earthworms were observed in the controls and at the application rates of 62.5, 125 and 500 mg/kg soil dry weight after 28 days of exposure. At the application rate of 250 mg/kg soil dry weight one earthworm showed yellow excretion from the oral aperture and at 1000 mg/kg soil dry weight two earthworms showed ulcer and skin bleedings. These minor pathological symptoms were not considered to be of biological significance.

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.

Any other information on results incl. tables

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	Mean
Control	0	0	0	0	0
Solvent Control	0	0	0	0	0
62.5	0	0	10	0	2.5
125	0	0	0	0	0
250	0	0	0	0	0
500	0	10	10	0	5
1000	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
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	9/10	10/10
	t)	¿	¿	1/10	¿
125	a)	10/10	10/10	10/10	10/10
250	a)	9/10	10/10	10/10	10/10
	f)	1/10	¿	¿	¿
500	a)	10/10	9/10	9/10	10/10
	t)	¿	1/10	1/10	¿
1000	a)	10/10	10/10	10/10	8/10
	g)	¿	¿	¿	2/10

a) no observation of symptoms or behavioural changes     f) yellow excretion from the oral aperture

g) ulcer and skin bleedings     t) dead earthworms     -) no observation

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	0.464	0.580	0.12	25.0	0.13 ± 0.01	29.0 ± 3.58	¿
	2	0.436	0.583	0.15	33.7
	3	0.451	0.586	0.14	29.9
	4	0.411	0.548	0.14	33.3
Solvent Control	1	0.445	0.583	0.14	31.0
	2	0.456	0.569	0.11	24.8
	3	0.414	0.529	0.12	27.8
	4	0.441	0.556	0.12	26.1
62.5	1	0.442	0.576	0.13	30.3	0.12 ± 0.02	27.3 ± 3.06	No
	2	0.410	0.505	0.10	23.2
	3	0.456	0.587	0.13	28.7
	4	0.448	0.569	0.12	27.0
125	1	0.411	0.561	0.15	36.5	0.14 ± 0.02	32.5 ± 5.35	No
	2	0.448	0.565	0.12	26.1
	3	0.440	0.572	0.13	30.0
	4	0.462	0.634	0.17	37.3
250	1	0.457	0.585	0.13	28.0	0.13 ± 0.01	30.0 ± 3.06	No
	2	0.443	0.562	0.12	26.9
	3	0.407	0.542	0.14	33.2
	4	0.449	0.593	0.14	32.1
500	1	0.451	0.567	0.12	25.7	0.14 ± 0.06	33.5 ±15.0	No
	2	0.464	0.548	0.08	18.1
	3	0.442	0.610	0.17	38.0
	4	0.405	0.617	0.21	52.3
1000	1	0.458	0.569	0.11	24.2	0.13 ± 0.04	29.6 ± 9.70	No
	2	0.443	0.531	0.09	19.9
	3	0.408	0.579	0.17	41.9
	4	0.452	0.598	0.15	32.3

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

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	398	367 ± 46.3	12.6	¿	¿
	2	365
	3	374
	4	399
Solvent Control	1	440
	2	343
	3	301
	4	315
62.5	1	408	356 ± 52.4	14.7	96.9	No
	2	283
	3	363
	4	368
125	1	352	349 ± 36.8	10.5	95.1	No
	2	397
	3	337
	4	309
250	1	307	352 ± 48.6	13.8	96.0	No
	2	401
	3	387
	4	314
500	1	469	385 ± 57.8	15.0	105	No
	2	374
	3	355
	4	341
1000	1	444	368 ± 57.4	15.6	100	No
	2	373
	3	307
	4	349

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the submission substance did not induce biologically significant 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 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) from June 10^thto August 6^th, 2009 at Dr.U.Noack-Laboratorienin 31157 Sarstedt , Germany . 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 exposure in soil, there were no noteworthy earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms across all treatments. The earthworm biomass increased in the control and at all application rates over 28 days without any statistically significant differences compared to the pooled control . After further four weeks the reproduction rate (average number of juveniles) was 367 in the control (control and solvent control) and ranged between 349 and 385 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.

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Tested Doses

Effects	Submission substance
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

Submission substance
	Application rate
	mg test item/kg soil dry weight
NOECreproduction, mortality, body weight	1000
LOECreproduction, mortality, body weight	> 1000