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Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

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

Adequacy of study:

key study

Study period:

From January 14 to February 05, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Details for read across approach are included into the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicais 202, Part II "Reproductiun Test at least 14 Days“, Paris/France

Version / remarks:

adopted Aprii 1984

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: samples of 20 ml were taken in duplicate of 2.5, 5 and 10 mg/l treated medium and of the control.
- Sampling time: samples were taken at the first and the last treatment period (last water renewal) directly after treatment and at the end of the respective period.
- Sampling method: the samples were taken from the middle of the test beakers. The test medium was not stirred before sampling.
- Sample storage conditions before analysis: the samples were submitted for immediate analyses.

Details on test solutions:

The test medium was treated with the test article before introduction of daphnids. Due to the dark colour of the substance in test medium, the highest possible test concentration was 10 mg/l. The appearance as well as the mobility was checked in a pre-test.
An amount of 100 mg test item was made up to 100 ml with test medium (1000 mcg/ml). The stock solution was freshly prepared at each treatment day.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: testing laboratory.
- Age of parent organisms: for the tests, animals with an age of less than 24 hours from about 3 week old adults were used.
- Feeding: feeding was performed daily by adding Scenedesmus subspicatus corresponding to 0.1 mg C/parent animal.

BREEDING
- Stock cultures: maintained in about 10 l glass vessels in mixtures of pond water and reconstituted water with slight aeration. During week one, 300-400 daphnids (<3 days old) were held in 10 l pond water; during week two, 250 of these daphnids with eggs in their brood pouch were held in a mixture of 1/3 reconstituted water and 2/3 pond water; during week three, these daphnids were held in a mixture of 1/3 pond water and 2/ 3 reconstituted water.
- Feeding: daphnids were fed with algae (Scenedesmus subspicatus or Chorella vulgaris or Selenastrum capicornutum) at least three times per week.
- Conditions: daphnids were kept under the same temperature and light conditions as in the test itself.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

the temperature in the untreated control vessels was measured using a thermometer. The temperature ranged from 19.3 °C to 21.0 °C at the end of the treatment periods

pH:

the initial pH of the systems was, on average, 7.9 for untreated, 7.8 and 7.9 for the treated samples. At the end of the exposure period, the pH for the respective concentrations were 8.4, 8.5 and 8.4

Dissolved oxygen:

the initiai oxygen concentration was, on average, 8.7 mg oxygen/l for untreated and for the treated samples. At the end of the exposure periods, the oxygen concentration of the respective samples were 8.3, 8.7 and 8.5 mg oxygen/l

Nominal and measured concentrations:

0.625, 1.25, 2.5, 5 and 10 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass 150 ml (80 × 55 mm) or 250 ml (95 × 75 mm).
- Aeration: none during the test.
- Renewal rate of test solution: the test medium (treated and untreated) was renewed at day 2, 5, 7, 9, 12, 14, 16 and 19 of the exposure period, every Monday, Wednesday and Friday.
- No. of organisms per vessel: glass-beakers each containing 1 Daghnia magna (less than 24 hours old) in at least 100 ml test medium.
- No. of organisms per concentration: 10 parent animals.
- No. of vessels per concentration: one replicate.
- No. of vessels per control: one replicate.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: the tests were performed in a mixture of 2/3 reconstituted water and 1/3 filtered pond water.
- pH: 7.6
- Hardness: 14.2 - 15.0 dH°
- Conducivity of mixture: 584 µS/cm.
- Aeration: before use, the test medium was aerated until oxygen saturation (8.2 - 9.4 mg O2/l).

OTHER TEST CONDITIONS
- Photoperiod: 16 hours/day
- Light intensity: 500 - 2000 Lux

EFFECT PARAMETERS MEASURED
The immobility of adults and the number of offspring were counted three times per week during transfer of adults in freshly treated test medium. Between day 19 and 21, counting was performed daily. Dead animals and offspring were removed.

MEASUREMENTS
- pH: the pH of the controls, the lowest (0.625 mg/l) and highest (10 mg/l) test concentrations of the test article was measured at all treatment periods at the beginning and end of the respective periods.
- Oxygen: the oxygen concentration should be > 60 % of the saturation concentration at the end of the treatment period (> 5 mg/l).

Reference substance (positive control):

yes

Remarks:

potassium dichromate, tested twice per year

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and mortality

Details on results:

With the exception of the concentration 2.5 mg/l, no immobility was observed at any concentrations. One daphnia at 2.5 mg/l had died between day 20 and 21.
Reproduction of young daphnids started between day 7 and 9 of the exposure period. At the end of the study, in total 1014, 1070, 1069, 1153, 1275 and 1128 offspring were produced by the 10 adult daphnids in the untreated, 0.625, 1.25, 2.5, 5 and 10 mg/l treated test medium, respectively.
No statistically significant influence of the test article on the reproduction rate was observed at 0.625, 2.5 and 10 mg/l. The observed significant effect at 1.25 and 5 mg/l is biologically not relevant, since higher reproduction rate than in the control was observed. Therefore, the NOEC and LOEC can be defined as equal/higher than 10 mg/l and higher than 10 mg/l, respectively.

CONTROLS
During the test, no immobility in the controls was observed.

MEASURED CONCENTRATIONS
The actual measured concentration was higher than 79.6 % of the nominal concentration. Therefore, all values are expressed as nominal concentration.

VALIDITY OF THE TEST
Any immobility was observed in the control. The total offspring of the control was, on average, 101 per animai. The coefficient of variation around the mean number of offsprings was 10 % in the control. Therefore, the test is valid.

Survival of parent daphnids exposed totest item at various concentrations in a semi-static system for 21 days.

Exposure day	Nominal conc. (mg/l)
	0	0.625	1.25	2.5	5	10
0	10	10	10	10	10	10
2	10	10	10	10	10	10
5	10	10	10	10	10	10
7	10	10	10	10	10	10
9	10	10	10	10	10	10
12	10	10	10	10	10	10
14	10	10	10	10	10	10
16	10	10	10	10	10	10
19	10	10	10	10	10	10
20	10	10	10	10	10	10
21	10	10	10	9	10	10
% survival	100	100	100	90	100	100

Influence of test item on the total number of live, young daphnids reproduced by the surviving adults (cumulative values).

Exposure day	Nominal conc. (mg/l)
	0	0.625	1.25	2.5	5	10
0	0	0	0	0	0	0
2	0	0	0	0	0	0
5	0	0	0	0	0	0
7	0	0	0	0	0	0
9	75	69	91	70	43	34
12	329	315	346	357	335	247
14	345	376	346	377	357	247
16	515	498	553	587	540	445
19	917	834	981	1039	991	848
20	962	999	981	1039	1056	848
21	1014	1070	1069	1153	1275	1128

Influence of test item on the reproduction rate of daphnids.

Replicae	Nominal conc. (mg/l)
	0^1	0.625	1.25	2.5	5	10
1	91 (0)	*0^2(0)	135 (0)	118 (0)	131 (2)	118 (0)
2	99 (0)	142 (0)	113 (0)	139 (0)	133 (0)	134 (0)
3	110 (0)	118 (0)	98 (0)	108 (0)	106 (0)	99 (0)
4	101 (0)	103 (0)	107 (0)	107 (0)	158 (0)	94 (0)
5	118 (0)	104 (0)	122 (0)	114 (0)	156 (0)	110 (0)
6	112 (0)	145 (0)	139 (0)	120 (0)	117 (0)	113 (0)
7	88 (0)	120 (0)	112 (0)	114 (0)	110 (0)	113 (0)
8	92 (0)	98 (0)	123 (0)	104 (0)	116 (0)	116 (0)
9	104 (0)	116 (0)	120 (0)	122 (0)	119 (0)	120 (0)
10	99 (0)	124 (0)	*0^2(0)	107 (1)	129 (0)	111 (0)
Mean ± s	101 ± 10	119 ± 16	119 ± 13	115 ± 11	128 ± 18	113 ± 11
In % of control	--	> 100	> 100	> 100	> 100	> 100
St	--	n.s.	n.s.	n.s.	n.s.	n.s.

In brackets: number of dead daphnids reproduced in addition.

St : Steel-test:

The steel-test (many-one rank test) was applied, since the data could not be assumed to follow a normal distribution.

1 Control.

2 No eggs produced.

* Not used for the statistic test and mean calculation.

n.s. No significant effect.

MEASURED CONCENTRATIONS

Nominal conc (mg/l)	Time (d)		Conc. of test item (mg/l)	Mean conc. samples A and B (mg/l)	% of nominal
	Start	End
Control	0/A		< 0.1	--	--
Control	0/B		< 0.1
Control		2/A	< 0.1	--	--
Control		2/B	< 0.1
Control	19/A		< 0.1	--	--
Control	19/B		< 0.1
Control		21/A	< 0.1	--	--
Control		21/B	< 0.1
2.5	0/A		2.271	2.253	90.1
2.5	0/B		2.235
2.5		2/A	2.765	2.715	108.6
2.5		2/B	2.665
2.5	19/A		2.019	2.011	80.5
2.5	19/B		2.002
2.5		21/A	2.481	2.44	97.6
2.5		21/B	2.399
5.0	0/A		4.372	4.409	88.2
5.0	0/B		4.446
5.0		2/A	5.162	5.072	101.4
5.0		2/B	4.982
5.0	19/A		4.032	4.023	80.5
5.0	19/B		4.015
5.0		21/A	4.579	4.733	94.7
5.0		21/B	4.887
10.0	0/A		8.851	8.879	88.8
10.0	0/B		8.906
10.0		2/A	10.12	10.13	101.3
10.0		2/B	10.14
10.0	19/A		7.952	7.961	79.6
10.0	19/B		7.969
10.0		21/A	9.103	9.422	94.2
10.0		21/B	9.74
Stock solution
1000.0	0/A		903.3	885.8	88.6
1000.0	0/B		868.3

A/B samples in duplicate

Conclusions:

NOEC (21d) ≥ 10 mg/l (nominal), based on mortality and reproduction

Executive summary:

The influence of test item on the reproduction and survival rate of Daghnia magna was investigated in a 21 day semi-static test, according to the OECD guideline 202, part II (1984). The test animals were exposed to nominal concentrations in the range 0.625 mg/l to 10 mg/l. The NOEC- and LOEC-values for reproduction rate were found to be equal/higher than 10 mg/l and higher than 10 mg/l, respectively. Both the EC50 value of the reproduction rate and the EC50 value for the immobility of the parental generation were found to be higher than 10 mg/l.

Conclusion

NOEC (21d) ≥ 10 mg/l (nominal), based on mortality and reproduction.

Description of key information

NOEC (21d) ≥ 10 mg/l (nominal), based on mortality and reproduction

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10 mg/L

Additional information

No studies on the long-term toxicity to aquatic invertebrates are available for the Direct Blue 267. Nevertheless, a study conducted on the structural analogue Similar Substance 01 is available; the read across approach can be considered reliable and appropriate to investigate the property (details for the approach are included into the IUCLID section 13).

The influence of Similar Substance 01 on the reproduction and survival rate of Daghnia magna was investigated in a 21 day semi-static test, according to the OECD guideline 202, part II (1984). The test animals were exposed to nominal concentrations in the range 0.625 mg/l to 10 mg/l. With the exception of the concentration 2.5 mg/l, no immobility was observed at any concentrations. One daphnia at 2.5 mg/l had died between day 20 and 21. Reproduction of young daphnids started between day 7 and 9 of the exposure period. No statistically significant influence of the test article on the reproduction rate was observed at 0.625, 2.5 and 10 mg/l. The observed significant effect at 1.25 and 5 mg/l is biologically not relevant, since higher reproduction rate than in the control was observed. Therefore, the NOEC and LOEC can be defined as equal/higher than 10 mg/l and higher than 10 mg/l, respectively.

During the test, no immobility in the controls was observed. The actual measured concentration was higher than 79.6 % of the nominal concentration, therefore, all values are expressed as nominal concentration.