Trisodium hydrogen [2-[[α-[[3-[[4-chloro-6-[ethyl[3-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulphophenyl]azo]benzyl]azo]-4-sulphobenzoato(6-)]cuprate(4-)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

no

Analytical monitoring:

yes

Remarks:

WoE 2: yes and WoE 3: no

Vehicle:

no

Remarks:

WoE 2 and WoE 3: no

Details on test solutions:

WoE 2: The test solution was prepared by dissolving 50 mg of the test chemical in 100 ml of ADaM’s media achieving a final test concentrations of 500 mg/l. Test chemical concentrations used for the study were 0, 7.5, 15, 30, 60, 120 mg/l, respectively.

WoE 3: The stock solution 150 mg/l was prepared by dissolving test chemical in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water.

Test organisms (species):

Daphnia magna

Details on test organisms:

WoE 2:
TEST ORGANISM
- Common name: Water flea
- Strain/clone: Eggs of Daphnia magna were obtained from MicroBio tests Kleimoer 15B-9030 MARIAKERKE (GENT) BELGIUM
- Feeding during test: no
- Other: A population of parthenogenetic females of synchronized age structure has been maintained for more than 2 years in the test facility under constant temperature conditions (18 to 22°C) at a 16 : 8 hour light-dark photoperiod (illumination: < 1000 lux). The culture media (Adams medium') was partly renewed once a week. During the maintenance of test organism, test daphnids were fed with unicellular green algae (Selenestrum capricornutum).

WoE 3:
TEST ORGANISM
- Common name: Water flea
- Strain: Straus
- Source: Own breeding at University of Chemistry and Technology, Prague
- Age at study initiation (mean and range, SD): The animals used for the test shall be less than 24 h old and should not be first brood progeny
- Feeding during test: No feeding

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

WoE 2: 140 mg of CaCO3

Test temperature:

WoE 2: 18-22°C
WoE 3: 20±1°C

pH:

WoE 2: no data available
WoE 3: without adjustment
sample at concentration 150 mg/l: pH = 8.1 changed to pH = 7.8 during the test
control: pH = 8.0 changed to pH = 7.7 during the test

Dissolved oxygen:

WoE 2: no data available
WoE 3: higher than 8.0 mg/L at the end of test both in the control and the sample

Nominal and measured concentrations:

WoE 2: Nominal concentrations: 0, 7.5, 15, 30, 60, 120 mg/l
WoE 3: Nominal test chemical concentration used for the study were 0, 30, 45, 67.5, 100 and 150 mg/l, respectively.

Details on test conditions:

WoE 2:
TEST SYSTEM
- Test vessel: Glass beaker
- Aeration: No aeration during experiment
- Material, size, headspace, fill volume: 25 ml of glass beaker filled with 20 ml media having headspace of 5 ml
- No. of organisms per vessel: 10 daphnids

OTHER TEST CONDITIONS
- Photoperiod:16 hours light and 8 hours dark
- Light intensity: 1000 – 1500 Lux

WoE 3:
TEST SYSTEM
- Test vessel: 50 ml glass vessel
- fill volume: 25 ml
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Natural water (surface or ground water), reconstituted water or dechlorinated tap water are acceptable as culturing and dilution water if D. magna survives in it for the duration of the culturing, acclimation and testing without showing signs of stress. Waters in the range pH 6 to pH 9, with hardness between 140 mg/l and 275 mg/l (as CaCO3) are recommended.
As an example, the preparation of dilution water meeting the requirements is described below.
Dissolve known quantities of reagents in water. The dilution water prepared shall have a pH of 7.8 ± 0.5, a hardness of (225 ± 50) mg/l (expressed as CaCO3), a molar Ca + Mg ratio close to 4 + 1 and a dissolved oxygen concentration above 7 mg/l.

Prepare the solutions specified below:
- Calcium chloride solution: Dissolve 117.6 g of calcium chloride dihydrate (CaCl2.2H2O) in water (4.2) and make up to 1 l with water (4.2).
- Magnesium sulfate solution: Dissolve 49.3 g of magnesium sulfate heptahydrate (MgSO4.7H2O) in water (4.2) and make up to 1 l with water (4.2).
- Sodium bicarbonate solution: Dissolve 25.9 g of sodium bicarbonate (NaHCO3) in water (4.2) and make up to 1 l with water (4.2).
- Potassium chloride solution: Dissolve 2.3 g of potassium chloride (KCI) in water (4.2) and make up to 1 l with water (4.2).

Mixing
Mix 2.5 ml of each of the four solutions and make up to 1 l with water.
The dilution water shall be aerated until the dissolved oxygen concentration has reached saturation and the pH has stabilized. If necessary, adjust the pH to 7.8 ± 0.5 by adding sodium hydroxide (NaOH) solution or hydrochloric acid (HCI). The dilution water prepared in this way shall not be further aerated before use.

- Sodium hydroxide solution, e.g. [NaOH] : 1 mol/l.
- Hydrochloric acid, e.g. [HCl] : 1 mol/l.

Reference substance:
Dissolve 600 mg of potassium dichromate (K2Cr2O7) in water and make up to 1 l with water (4.2).

OTHER TEST CONDITIONS
- Adjustment of pH: no adjustment done
- Photoperiod: No - Darkness
- Light intensity:

CALCULATION:
EC50 was calculated using non linear regression by the software Prism 4.0

Reference substance (positive control):

yes

Remarks:

WoE 2 and WoE 3: potassium dichromate was used as a reference substance for the study.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: WoE 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

103.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: WoE 3: 95 % CI. = 77.2 to 139.6 mg/l

Results with reference substance (positive control):

WoE 2: The 48 hr EC50 value of reference substance potassium dichromate was determined to be 0.831 mg/l.
WoE 3: - Results with reference substance valid
- EC50: 0.73 mg/L (24 hours)

Reported statistics and error estimates:

WoE 2: no data available
WoE 3: EC50 was calculated using non linear regression by the software Prism 4.0.

WoE 2:

Table: Assessment of test concentrations

Sr. no.	Concentrations (mg/l)	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	612	0.00	25
2	5.00	612	0.07	25
3	10.00	612	0.14	25
4	20.00	612	0.29	25
5	30.00	612	0.43	25
6	40.00	612	0.57	25
7	50.00	612	0.71	25
8	60.00	612	0.84	25
9	70.00	612	1.03	25
10	80.00	612	1.17	25
11	90.00	612	1.29	25
12	100.00	612	1.43	25
13	110.00	612	1.53	25
14	120.00	612	1.64	25

 

The absorbance and concentrations were recorded at 202 nm.

Table: Concentration after analytical Determination

Sr. No	Concentrations (mg/L)	Absorbance (mean) (0 hour)	Analytical Concentrations (0 hour)	Absorbance (mean) (48 hour)	Analytical Concentrations (48 hour)
1	blank	0.00	0.00	0.00	0.00
2	7.5	0.10	7.74	0.12	9.00
3	15	0.22	16.26	0.25	17.70
4	30	0.44	31.72	0.47	33.90
5	60	0.87	61.94	0.93	65.87
6	120	1.56	110.63	1.61	114.60

 

Table: pH, DO AND TEMPERATURE

Test Concentration (mg/L)	pH			Dissolved oxygen			Temperature °C
	0 Hour	24 Hour	48 Hour	0 Hour	24 Hour	48 Hour	0 Hour	24 Hour	48 Hour
Control	7.9	7.9	7.6	7.8	6.9	5.2	20.0	19	20
7.5	7.9	7.5	7.7	7.5	6.8	5.0	20.0	19	20
15	7.8	7.5	7.4	7.5	6.6	5.1	20.0	19	20
30	7.9	7.4	7.4	7.5	6.8	4.9	20.0	19	20
60	7.9	7.8	7.3	7.6	6.5	5.0	20.0	19	20
120	7.8	7.5	7.2	7.5	6.6	4..8	20.0	19	20

 

 

Table: IMMOBILIZATION AND INHIBITION

Sr. No	Test Concentrations (mg/L)	0 Hour	24 Hour	48 Hour	Cumulative Immobilization	Percent Inhibition
1	Control	-	-	-	-	-
2	7.5	-	-	-	-	-
3	15	-	-	-	-	-
4	30	-	-	-	-	-
5	60	-	-	-	-	-
6	120	-	-	-	-	-
		No abnormality observed	All daphnids are alive	No immobility observed

 

WoE 3:

In control solution containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test.

Result of the definitive test:

Sample no.	Sample Information	Conc. (mg/l)	I%	48 hr EC50
				mg/l	95% C. I. (mg/l)
Control	Reconstituted water	0	0
Test chemical	Test chemical	30.0 45.0 67.5 100.0 150.0	5 20.0 40.0 50.0 60.0	103.8	77.2 to 139.6

Validity criteria fulfilled:

yes

Remarks:

1. In the control, including the control containing the solubilising agent, not more that 10 percent of the daphnids should have been immobilized. 2. The dissolved oxygen concentration at the end of the test should be 3 mg/l in control and test vessels

Conclusions:

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism Daphnia magna, the 48hr EC50 value can be expected to be > 100 mg/l. Thus, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per CLP classification criteria.

Executive summary:

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic invertebrates. The studies are as mentioned below:

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The test solution was prepared by dissolving 50 mg of the test chemical in 100 ml of ADaM’s media achieving a final test concentrations of 500 mg/l. Test chemical concentrations used for the study were 0, 7.5, 15, 30, 60, 120 mg/l, respectively. Test concentrations were verified analytically by UV-Vis Spectrophotometer. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals were exposed to medium (i.e.a beaker containing only medium) and the test chemical concentrations for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 48 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 120 mg/L. Thus, based on the EC50 value, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

Another short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 150 mg/l was prepared by dissolving test chemical in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Nominal test chemical concentrations used for the study were 0, 30, 45, 67.5, 100 and 150 mg/l, respectively. Study was performed using 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 103.8 mg/l (95 % CI. = 77.2 to 139.6 mg/l). Thus, test chemical was considered as non-toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism Daphnia magna, the 48hr EC50 value can be expected to be > 100 mg/l. Thus, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per CLP classification criteria.

Description of key information

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism Daphnia magna, the 48hr EC50 value can be expected to be > 100 mg/l. Thus, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per CLP classification criteria.

Additional information

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic invertebrates. The studies are as mentioned below:

 

An acute immobilisation test was conducted for 48 hrs for assessing the effect of test chemical on Daphnia magna. The test was performed in accordance to OECD guideline No. 202“Daphnia sp.,Acute Immobilization Test”. The test solution was prepared by dissolving 50 mg of the test chemical in 100 ml of ADaM’s media achieving a final test concentrations of 500 mg/l. Test chemical concentrations used for the study were 0, 7.5, 15, 30, 60, 120 mg/l, respectively. Test concentrations were verified analytically by UV-Vis Spectrophotometer. Study was performed using 10 daphnids in a static system. Total 10 Daphnids/conc. were exposed to test chemical in 25 ml beakers in a volume of 20 ml of liquid solution containing both the chemical and media. The beakers were placed in a room at a temperature of 18 -22°C, hardness of water 140 mg of CaCO3 and under a photoperiod of 16:8 hr light: dark conditions with light intensity 1000 – 1500 Lux, respectively. One control vessel was also run simultaneously during the study. The animals were exposed to medium (i.e.a beaker containing only medium) and the test chemical concentrations for a period of 48 hour. Potassium dichromate was used as a reference substance for the study. The 48 hr EC50 value of reference substance was determined to be 0.831 mg/l. No Immobility were found in the control test animals and the dissolved oxygen concentration at the end of the test in the control and test vessel was ≥ 3 mg/l, thus validity criterion of the study has been fulfilled. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of effect of test chemical on mobility of the test organism, the median effect concentration (EC50 (48 h)) value was determined to be > 120 mg/L. Thus, based on the EC50 value, chemical was considered as non-toxic to aquatic invertebrates and hence, considered to be 'not classified' as per CLP classification criteria.

 

Another short term toxicity to aquatic invertebrate study was conducted for 48 hrs for assessing the effect of test chemical. The test was performed in accordance to OECD guideline No. 202 “Daphnia sp.,Acute Immobilization Test”. Daphnia magna was used as a test organism for the study. The stock solution 150 mg/l was prepared by dissolving test chemical in reconstituted water. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with reconstituted test water. Nominal test chemical concentrations used for the study were 0, 30, 45, 67.5, 100 and 150 mg/l, respectively. Study was performed using 5 organisms per vessel/replicates in a static fresh water system. Daphnids were exposed to test chemical in 50 ml glass vessel in a volume of 25 ml of liquid solution containing both the chemical and media. Control solution vessel containing reconstituted water without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 20±1°C. With the test substance one positive control Potassium dichromate (K2Cr2O7) was run simultaneously. EC50 was calculated using non linear regression by the software Prism 4. In the control vessel containing reconstituted water without the test chemical, no daphnids were immobilized at the end of the test. On the basis of the mobility of the test organism Daphnia magna due to the exposure of test chemical, the 48hr median effect concentration (EC50) value was determined to be 103.8 mg/l (95 % CI. = 77.2 to 139.6 mg/l). Thus, test chemical was considered as non-toxic to aquatic invertebrates at environmental related concentrations and hence, considered to be 'not classified' as per the CLP classification criteria.

 

On the basis of the experimental studies of the read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism Daphnia magna, the 48hr EC50 value can be expected to be > 100 mg/l. Thus, test chemical was considered as non-toxic and hence, considered to be 'not classified' as per CLP classification criteria.