1-hydroxy-4-[[4-[(methylsulphonyl)oxy]phenyl]amino]anthraquinone

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes

Specific details on test material used for the study:

Physical appearance: Black powder
Purity as per CoA: 99.1 % (w/w)
Batch No.: 1404301
Manufactured date: 20 September 2014
Expiry date: 08 April 2024
Density: 1.2654 g/cm³
pH: 5.0 (susp. of 2 % (w/w) in water)
Recommended storage condition: Ambient (+15 to +25 °C)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

Test Item Solubility
The test item was tested for its solubility in acetone and this test item mixture formed suspension after introduction into Elendt M4 medium at 0.1 mg/mL (performed as pre-study).

Test Medium Preparation and Renewal
Test solutions of the chosen concentrations were prepared by dilution of a stock solution. Stock solution was prepared using test medium using
sonication until uniform formulation was obtained.
Based on the results of preliminary stability tests, test medium was renewed once in 48 h (2 days) depending on the stability (found to be stable for 72 h) of the test substance in the test medium.
When the medium was renewed during semi-static test (± 1 h from treatment start on day 0 exposure), a second series of test vessels were prepared and the parent animals transferred to them by a dedicated pipette. The volume of medium transferred with the Daphnia was minimal.

Test organisms (species):

Daphnia magna

Details on test organisms:

Daphnia magna was used as the test system as it is a recommended species in the guideline. Daphnia magna obtained from the following source was used in this test and stock culture (Ecotoxicology, Eurofins Advinus limited, Bengaluru, India) maintained as below:
Eurofins Agroscience Services Ecotox GmbH
Aquatic Toxicology, Eutinger Strasse 24
D-75223 Niefern Öschelbronn, Germany
Daphnia were maintained in dilution medium (like one which was used in the test for acclimatization as well as preparation of test medium). The test medium in which the Daphnia were maintained is changed at least twice in a week and they are fed with the alga, Pseudokirchneriella subcapitata.

Neonates (Daphnids < 24 hour old)
At the start of the test, the animals were less than 24 hours old and were not first brood progeny. They were derived from a healthy stock (i.e., showing no signs of stress such as high mortality, presence of males and ephippia, delay in the production of the first brood, discoloured animals, etc.). The stock animals were maintained in culture conditions (light, temperature, medium, feeding and animals per unit volume) similar to those used in this test.
Adult gravid daphnids (which are already given their broods) were incubated individually in 100 mL of test medium, a day prior to start of the treatment.
Progenies produced were observed (on the day of treatment) for neonate sex and after ascertaining the exclusive production of female progenies then the treatment was planned. Female daphnids aged less than 24 hours were used in the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Hardness was 250 mg/L as CaCO3 (measured using commercially available strips)

Test temperature:

The test medium temperature was between 19.7 °C and 19.9 °C during the range finding test.
The test medium temperature was between 19.4 °C and 19.7 °C during the definitive test.

pH:

The pH was within the range 6.05 - 6.92 during the range finding test.
The pH was within the range 6.08 - 6.84 during the definitive test.

Nominal and measured concentrations:

Time-weighted mean test concentrations are 0.03, 0.06, 0.19, 0.67 and 2.23 mg/L (respective nominal test concentrations are 0.06, 0.18, 0.54, 1.62
and 4.86 mg/L).

Details on test conditions:

Test Conditions
Female daphnids were exposed to the test concentrations along with a control and vehicle control under the following conditions (range finding and definitive tests). Incubator was used for incubating the test vessels during the test.

Test containers : Glass beakers of 250 mL capacity were used as test vessels
Method of exposure : Semi-static (renewal of test medium at every 48 h interval)
Duration of the test : 21 days
No. of Daphnia : 2 Daphnids held individually per group during the range finding test (2 replicates)
10 Daphnids held individually per group during the definitive test (10 replicates)
Loading : 100 mL test medium in each replicate
Light intensity : 1200 - 1290 lux at the level of test vessel during the range finding test
1270 - 1390 lux at the level of test vessel during the definitive test
Light : Cool white light of 16-hour and 8-hour dark cycles
Aeration : The test medium was not aerated during the test
Feeding : Feeding was made daily and the possible dilution of the exposure concentrations by food addition was considered and avoided as much as possible with well concentrated algae suspensions. The diet of the parent animals was living algal cells (20 μL) of Pseudokirchneriella subcapitata. The supplied diet was based on the amount of organic carbon (C) provided to each parent animal. Ration level of 0.2 mg C/Daphnia/day was provided for each parent animal

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Results

Test Item Solubility

The test item formed suspension after introduction into acetone and Elendt M4 medium at 0.1 mg/mL (performed as pre-study).

Range Finding Test

Range finding test resulted in toxic effects on survival and reproduction of daphnids.

Concentration (mg/L)	Parent mortality (%) out of 2	Average offspring produced per parent animal	%RO
G1-Control	0	83.0	-
G2-Vehicle control	0	82.5	-0.6
G3-0.1	0	83.5	0.6
G4-1	50	71.0	-14.5
G5-10	100	41.0	-50.6
G6-50	100	0.0	-100.0
G7-100	100	0.0	-100.0

%RO: % Reduction in offspring production

Male neonates and ephippia were not observed during the test. There were no instances of aborted eggs, aborted broods or dead offspring (except in G4 and G5) in the control, vehicle control and any of the test item treated groups.

Definitive Test

There was no mortality of parent animals in the control and vehicle control. The mortality of parent animals was 0, 0, 0, 60 and 100 % on day 21 of exposure at the test concentrations of 0.03, 0.06, 0.19, 0.67 and 2.23 mg/L (respective nominal test concentrations are 0.06, 0.18, 0.54, 1.62 and
4.86 mg/L), respectively.

The percent change in reproductive output of parent animals was 0.4, -28.1, -38.4, -46.5 and -61.0 % at the test concentrations of 0.03, 0.06, 0.19, 0.67 and 2.23 mg/L, respectively. Reproductive output of parent animals evidenced statistically significant reduction in the tested concentrations of 0.06, 0.19, 0.67 and 2.23 mg/L as compared with the vehicle control group. Analysis of the number of living offspring per surviving parent animal also evidenced statistically significant reduction in the tested concentrations of 0.06, 0.19 and 0.67 mg/L as compared with the vehicle control group.

Time to production of first brood (and subsequent broods), number and size of broods evidenced prolongation (production of broods) and reduction (number and size of broods) in the tested concentrations of 0.06, 0.19, 0.67 and 2.23 mg/L as compared with the vehicle control group.

The percent production of male offspring was 0% in all the test concentrations of 0 (control), 0 (vehicle control), 0.03, 0.06, 0.19, 0.67 and 2.23 mg/L.

Measurement of the length of the parent animals at the end of the test (day 21) evidenced statistically significant reduction in the tested concentrations of 0.06, 0.19, 0.67 and 2.23 mg/L as compared with the vehicle control group.

Intrinsic rate of population increase evaluation evidenced reduction at the tested concentrations of 0.06, 0.19, 0.67 and 2.23 mg/L as compared with the vehicle control. Abnormal and dead neonates were excluded during determination of intrinsic rate of population increase to obtain actual values.  

Male neonates and ephippia were not recorded during the test. There were no instances of aborted eggs, aborted broods or dead offspring (except in G5 to G7) in the control, vehicle control and any of the test item treated groups.

Reproduction endpoints were determined as total living offspring per maternal organism (parent animal) which did not die accidentally or inadvertently during the test.

The effects of FAT 36038/J TE on parent animals (Daphnia magna) mortality, growth effects (length of the parent animals) and reproduction based on nominal and time-weighted mean test concentrations are as below:

Endpoints		Nominal conc. (mg/L)	*Measured conc. (mg/L)	95% Fiducial Limits (mg/L)
				Lower	Upper
Mortality	NOEC	0.54	0.19	-	-
	LOEC	1.62	0.67	-	-
	LC50	1.592	0.6564	-	-
Growth effects	NOEC	0.06	0.03	-	-
	LOEC	0.18	0.06	-	-
Reproduction	NOEC	0.06	0.03	-	-
	LOEC	0.18	0.06	-	-
	EC10	0.03886	0.01337	0.0003451 (0.002894)	0.07074 (0.1576)
	EC20	0.1616	0.06027	0.007218 (0.03436)	0.1912 (0.4213)
	EC50	1.846	0.7908	0.3364 (0.9251)	3.634 (5.404)

*: Based on time-weighted mean test concentrations. Values in parentheses are fiducial limits based on nominal test concentrations in mg/L.

Analytical Verification of Test Item Concentration in Test Medium

The stability test results concluded that the test item was stable in the Elendt M4 medium at 72 h in the nominal concentrations of 0.0510 and 200.400 mg/L.

The results of test item analysis of test concentrations of 0.06, 0.18, 0.54, 1.62 and 4.86 mg/L showed that overall percent agreement was as below:

Sample	Mean Minimum (%)	Mean Maximum (%)
0 h Fresh	50.97	106.25
48 h Aged	7.42	35.17

Since the deviation from the measured initial concentration was greater than ± 20%. Hence, the results were expressed in terms of the time-weighted mean. Time-weighted mean test concentrations are 0.03, 0.06, 0.19, 0.67 and 2.23 mg/L (respective nominal test concentrations are 0.06, 0.18, 0.54, 1.62 and 4.86 mg/L). The analytical measurements of FAT 36038/J TE in test medium varied in random fashion. In most occasions, the analytical measurements were more than ± 20% from the nominal concentration during the test. This variation in analytical measurements doesn’t have any impact on the outcome of the test, since test concentrations are based on time-weighted mean measured concentrations (which provide exposure scenario of test item).

CONDITIONS FOR THE Validity of the Test

This test has met all the validity criteria in the control(s):

• The mortality of the parent animal (female daphnia) was 0% (control and vehicle control) at the end of the test (acceptable criterion: mortality of the parent animal should not exceed 20% at the end of the test).
• The mean number of live offspring produced per parent animal surviving at the end of the test is ≥ 79.8 and 80.3 in the control and vehicle control, respectively (acceptable criterion: ≥ 60).
• Maximum male offspring produced was 0 % in the control and vehicle control (acceptable criterion: no more than 5 % male offspring).
• The accidental and inadvertent parental mortality for the control(s) as well as for each of the test concentration was 0 % (acceptable criterion: should not exceed 20 % at the end of the test).

Validity criteria fulfilled:

yes

Conclusions:

The EC10 for reproduction in Daphnia magna when exposed for 21 days was determined to be 0.01337 mg/L.

Executive summary:

The reproductive effects of the test item, FAT 36038/J TE were studied on the reproductive output of daphnids, Daphnia magna. This test was conducted as a semi-static test with renewal of test medium at every 48 h interval. Mortality, behaviour and offspring produced (neonate sex) of the parent animals were observed daily during the 21 days test.

A range finding test was carried out to determine the test concentrations for the definitive test. Female, Daphnia magna aged less than 24 hours old were exposed to the nominal test item concentrations of 0.1, 1, 10, 50 and 100 mg/L along with control and vehicle control.

Range finding test resulted in toxic effects on survival and reproduction of daphnids, based on which the definitive test was conducted. In definitive test, Daphnia magna (female) less than 24 hours old were exposed to nominal test item concentrations of 0.06, 0.18, 0.54, 1.62 and 4.86 mg/L along with control and vehicle control.

The effects of FAT 36038/J TE on parent animals (Daphnia magna) mortality, growth effects (length of the parent animals) and reproduction based on nominal and time-weighted mean test concentrations are as below:

  

Endpoints		Nominal conc. (mg/L)	*Measured conc. (mg/L)	95% Fiducial Limits (mg/L)
				Lower	Upper
Mortality	NOEC	0.54	0.19	-	-
	LOEC	1.62	0.67	-	-
	LC50	1.592	0.6564	-	-
Growth effects	NOEC	0.06	0.03	-	-
	LOEC	0.18	0.06	-	-
Reproduction	NOEC	0.06	0.03	-	-
	LOEC	0.18	0.06	-	-
	EC10	0.03886	0.01337	0.0003451 (0.002894)	0.07074 (0.1576)
	EC20	0.1616	0.06027	0.007218 (0.03436)	0.1912 (0.4213)
	EC50	1.846	0.7908	0.3364 (0.9251)	3.634 (5.404)

*: Based on time-weighted mean test concentrations. Values in parentheses are fiducial limits based on nominal test concentrations in mg/L.

Description of key information

The EC10 for reproduction in Daphnia magna when exposed for 21 days was determined to be 0.01337 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

0.013 mg/L

Additional information

The reproductive effects of the test item, FAT 36038/J TE were studied on the reproductive output of daphnids, Daphnia magna. This test was conducted as a semi-static test with renewal of test medium at every 48 h interval. Mortality, behaviour and offspring produced (neonate sex) of the parent animals were observed daily during the 21 days test.

A range finding test was carried out to determine the test concentrations for the definitive test. Female, Daphnia magna aged less than 24 hours old were exposed to the nominal test item concentrations of 0.1, 1, 10, 50 and 100 mg/L along with control and vehicle control.

Range finding test resulted in toxic effects on survival and reproduction of daphnids, based on which the definitive test was conducted. In definitive test, Daphnia magna (female) less than 24 hours old were exposed to nominal test item concentrations of 0.06, 0.18, 0.54, 1.62 and 4.86 mg/L along with control and vehicle control.

The effects of FAT 36038/J TE on parent animals (Daphnia magna) mortality, growth effects (length of the parent animals) and reproduction based on nominal and time-weighted mean test concentrations are as below:

  

Endpoints		Nominal conc. (mg/L)	*Measured conc. (mg/L)	95% Fiducial Limits (mg/L)
				Lower	Upper
Mortality	NOEC	0.54	0.19	-	-
	LOEC	1.62	0.67	-	-
	LC50	1.592	0.6564	-	-
Growth effects	NOEC	0.06	0.03	-	-
	LOEC	0.18	0.06	-	-
Reproduction	NOEC	0.06	0.03	-	-
	LOEC	0.18	0.06	-	-
	EC10	0.03886	0.01337	0.0003451 (0.002894)	0.07074 (0.1576)
	EC20	0.1616	0.06027	0.007218 (0.03436)	0.1912 (0.4213)
	EC50	1.846	0.7908	0.3364 (0.9251)	3.634 (5.404)

*: Based on time-weighted mean test concentrations. Values in parentheses are fiducial limits based on nominal test concentrations in mg/L.