6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-01-19 to 1994-02-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD 202, Part II

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples (each comprising approximately 200 mL) were received on 8 occasions (19 January to 9 February 1994). One sample from each pair was used in the analysis, the remaining sample being stored at 4 °C in case a check analysis was required.

Vehicle:

yes

Details on test solutions:

- Concentration of vehicle in test medium: The test substance was dispersed in Tween 80 to give an initial stock paste of 50 mg/g.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia Magna Straus
- Age of parental stock (mean and range, SD): Gravid adults were isolated 24 hours prior to Initiation of the test. Young daphnids produced overnight were used for testing.
- Feeding during test: Yes
- Food type: mixed unicellular algal culture
- Amount: 5 mL
- Frequency: daily

ACCLIMATION
- Acclimation conditions: same as test
- Type of food: Suspension of mixed algae (predominantly Scenedesmus and Selenastrum spp).
- Feeding frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

350 mg CaCO3/L

Test temperature:

21 ± 1 °C

pH:

7.7

Dissolved oxygen:

7.8 - 8.1

Nominal and measured concentrations:

Nominal test concentrations: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
Measured test concentrations: 0.012, 0.030, 0.096, 0.25 and 0.63 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: flask
- Type: open
- Material, fill volume: glass, 400 mL
- Aeration: No
- Renewal rate of test solution: three times per week (Monday, Wednesday and Friday)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 medium was prepared using analytical grade reagents and reverse osmosis purified water (see "Any other information on materials and methods").
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light : 8 hours dark

EFFECT PARAMETERS MEASURED
The live and dead Daphnia of the "parental" (P) generation were counted daily and the numbers recorded together with observations on the general condition and size of the Daphnia as compared with the controls. The numbers of live and dead "filial" (F) Daphnia, the numbers of "parental" (P) Daphnia with eggs or young in the brood pouch, and the numbers of discarded unhatched eggs were counted and recorded immediately prior to test media renewal (ie three times per week).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125, 3.2

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.53 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: EC50 value far in excess of water solubility

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.53 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- There were no indications of acute or prolonged effects on the parental generation at concentrations well in excess of the water solubility value.
- Prolonged exposure of Daphnia magna to the test item resulted in a slight reduction in the numbers of young produced at the three highest exposure levels (0.045, 0.17 and 0.53 mg/L).
% loss in concentration over the test period: Mean measured concentrations for each dose level, as % of nominal, ranged from 32 to 53 %. Average loss was approximately 47 %.
The NOEC for immobility was recorded as >0.53 mg/L based on the measured concentrations. The NOEC for reproduction was statistically calculated as 0.012 mg/L using the Williams test with a lowest effect concentration of 0.045 mg/L. The EC10 was calculated to 0.027 mg/L.
No other adverse effects on survival or reproduction were observed. It should be recognized that the concentrations required to cause impairment of reproduction are unlikely to occur in the environment due to the low solubility of the test item in water. The "NOEC" based on measured concentrations approximates the water solubility value of the test substance.

Table 2: Validation Criteria

	Required	Actual
Surfactant control	≤ 20 %	15 %
Dissolved oxygen	≥ 60 % ASV	72 % ASV
pH (Surfactant control)	Deviation ≤ 0.3	0.3
First young (Surfactant control group)	Produced within 9 days	7 days
Cumulative young per female (control group)	≥ 40	40
Number of broods per solvent control group	≥ 3	≥ 3*
* Pooling of data from the 4 replicates tends to mask the identification of distinct broods but at least 3 broods are considered to have been produced given the numbers of young counted and the length of the reproductive phase in relation to the typical breeding pattern of Daphnia magna ASV Air Saturation Value

Table 3: Summary of the results

Nominal concentration mg/L	Measured concentration mg/L	% Survival of P1	No. live young		No. dead young		No. unhatched eggs
			Total	Per ♀	Total	Per ♀	Total	Per ♀
Control	Control	80	1289	40	0	0	17	<1
Surfactant control	Surfactant control	85	1376	40	3	<1	1	<1
0.010	0.012	75	1244	36	3	<1	19	<1
0.032	0.030	80	1208	37	1	<1	5	<1
0.10	0.096	80	1323	37	3	<1	21	<1
0.32	0.25	68	1294	41	1	<1	15	<1
1.0	0.63	73	1281	39	3	<1	48	~2

Validity criteria fulfilled:

not specified

Conclusions:

In a long-term toxicity study in Daphnia Magna accoridng to OECD guideline 202, a NOEC of above 0.63 mg/L was estimated.

Executive summary:

The long term aquatic toxicity to daphnia was examined according to OECD guideline 202, part II (Huntingdon, 1992). Water flea (daphnia magna) were exposed to the test substance at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L (measured: 0.012, 0.030, 0.096, 0.25 and 0.63 mg/L) under semi-static conditions. Continuous flow conditions could not be used due to the poor solubility of the test substance in water. Media was renewed at 48 - 72 hour intervals.

No significant mortalities occurred in Daphnia magna exposed to the test item for 21 days. Consequently it was not possible to calculate EC50 (immobilisation) values. The "no effect" level (immobilisation < 20 %) at 21 days > 0.63 mg/L. Background mortalities ranged from 15 -20 % and it is thought, therefore, that the 27 % immobilisation obtained at the highest test concentration is not significantly different. Higher test concentrations could not be prepared owing to the limited solubility of the test item in water (0.01 mg/L) and having regard to the amount of auxiliary surfactant permitted in the test.

The 14- and 21-day EC50 (reproduction) values have been estimated to be greater than 0.63 mg/L. Since the EC50 values are so far in excess of the water solubility value, meaningful EC50 values cannot be quoted. There were no other apparent differences in size, activity or any other characteristics between Daphnia of the control and treatment groups.

Information on the effects of the test item on the F1 generation is limited since, by study design, the young are removed soon after liberation from the brood pouch. Numbers of unhatched eggs and dead young were low in all control and treatment groups (<2 per female). The "no-observed effect concentration" (21 days) has been estimated to be >0.63 mg/L.

No adverse effects on survival or reproduction were observed in Daphnia magna exposed to the test item for 21 days. It should be noted that concentrations required to cause impairment of reproduction are unlikely to occur in the environment due to the low solubility of the test item in water (0.01 mg/L).

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-06-03 to 1991-06-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 202, Part II

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: solvent control and each test concentration (replicates pooled).
- Sampling method: Water samples (200 mL in duplicate) were taken from the solvent control and each test concentration (replicates pooled) on Days 0 (fresh media) 2, 7, 9, 14, 16, 18 and 21 (expired media)

Vehicle:

yes

Remarks:

Preliminary Solution in 1 % Tween 80-acetone.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: STRAUS
- Source: Laboratory culture orlginating from a strain supplied by IRChA, France.
- Age of parental stock: Gravid adults were isolated 24 hours prior to Initiation of the test. Young daphnids produced overnight were used for testing.
- Feeding during test: Yes
- Food type: Mixed unicellular algal culture supplemented with fry fish food (Liquifiry®).
- Amount: approximately 5 mL
- Frequency: daily

ACCLIMATION
- Acclimation conditions: same as test
- Type and amount of food: fry fish food (Liquifiry®) and a Suspension of mixed algae (predominantly Scenedesmus or Selenastrum spp.).
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): No

Test type:

other: 3 times per week (Days 2, 4, 7, 9, 11, 14, 16 and 18). More frequent renewal of test media would increase disturbance to the brood animals and increase the likelihood of impaired survival and reproduction.

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

160 mg/L (Ca)

Test temperature:

21 ± 1 °C

pH:

8.0 - 8.3

Dissolved oxygen:

8.0 - 8.6 mg/L

Nominal and measured concentrations:

Nominal: 0.010, 0.032, 0.10, 0.32, 1.0 mg/L
Measured: 0.032, 0.012, 0.045, 0.17, 0.53 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Flask
- Type: open
- Material, fill volume: Glass, 400 mL
- Aeration: None. The diluent only was aerated prior to test media preparation.
- Renewal rate of test solution 3 times per week (Days 2, 4, 7, 9, 11, 14, 16 and 18). More frequent renewal of test media would increase disturbance to the brood animals and increase the likelihood of impaired survival and reproduction.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated (with sodium thiosulphate) and aged laboratory tap water.
- Total organic carbon: 3.9 mg/L
- Metals:
Magnesium 8.8 mg/L
Sodium 58 mg/L
Potassium 10.0 mg/L
Calcium 130 mg/L
Aluminum 6.4 µg/L
Iron 110 µg/L
Manganese 20 µg/L
Copper 25 µg/L
Zinc 5.3 µg/L
Fluoride 260 µg/L

- Pesticides: 0.27 µg/L
- Chlorine: 0.26 mg/L
- Alkalinity: 150 mg/L (HCO3)
- Ca/Mg ratio: 14.77
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light : 8 h dark.

EFFECT PARAMETERS MEASURED
The live and dead Daphnia of the "parental" (P) generation were counted daily and recorded together with observations on the general condition and size of the Daphnia as compared with the controls. At each test media renewal the numbers of live and dead "filial" (F) Daphnia were recorded together with the number "parental" (P) Daphnia with eggs or young in the brood pouch. The number of discarded unhatched eggs was also determined at this time.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125 / 3.2
- Test concentrations:
Nominal: 0.010, 0.032, 0.10, 0.32, 1.0 mg/L
Measured: 0.032, 0.012, 0.045, 0.17, 0.53 mg/L

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 0.53 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Higher test concentrations could not be prepared owing to the limited solubility of the test item in water (0.01 mg/L) and having regard to the amount of auxiliary solvent permitted in the test.

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.027 mg/L

Nominal / measured:

estimated

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: This estimate was obtained in the Williams' test after comparison of data from treatment and solvent control groups.

Details on results:

- Behavioural abnormalities: None observed
- Mortality of control: 2 %
- Other adverse effects control: None observed
- Effect concentrations exceeding solubility of substance in test medium: Yes

Table 2: Summary 21-day exposure

Test concentration mg/L (nominal)	Test concentration mg/L (measured)	% survival of P1	No of live young		No of dead young		No of unhatched eggs
			Total	Per ♀	Total	Per ♀	Total	Per ♀
Control	Control	89	1619	42	0	0	0	0
Solvent control	Solvent control	93	1647	44	1	<1	2	<1
0.010	0.0032	93	1629	43	0	0	0	0
0.032	0.012	95	1533	40	0	0	1	<1
0.10	0.045	90	1388	37	0	0	0	0
0.32	0.17	93	1388	38	0	0	1	<1
1.0	0.53	93	1205	33	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

In an reproduction study in Daphnia magna according to OECD guideline 202, part 2, an EC50 (immobilisation and reproduction) of above 0.53 was determined. The EC10 for reproductive effects was calculated to be 0.027 mg/L. The NOEC for immobility is above 0.53 mg/L (nominal).

Executive summary:

The effect of the test item on the reproduction of Daphnia magna was assessed in a study according to OECD Guideline 202, part 2. Water flea (Daphnia magna) were exposed to the test substance at nominal concentrations of 0.01, 0.032, 0.1, 0.32 and 1.0 mg/L (measured 0.0032, 0.012, 0.045, 0.17 and 0.53 mg/L) under semi-static conditions. Media was renewed every 3 days. There were no indications of acute or prolonged effects on the parental generation at concentrations well in excess of the water solubility value. Prolonged exposure of Daphnia magna to the test item resulted in a slight reduction in the numbers of young produced at the three highest exposure levels (0.045, 0.17 and 0.53 mg/L). No other adverse effects on survival or reproduction were observed. It should be recognized that the concentrations required to cause impairment of reproduction are unlikely to occur in the environment due to the low solubility of the test item in water. The calculated "EC10" based on measured concentrations approximates the water solubility value of the test substance.

The NOEC for immobility was recorded as above 0.53 mg/L based on the measured concentrations. The EC10 for reproduction was statistically calculated as 0.027 mg/L using the Williams test with a lowest effect concentration of 0.045 mg/L.

Reference 3

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-01-17 to 1994-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 202, Part II

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Duplicate samples (each comprising approximately 200 mL) were received on 8 occasions (19 January to 9 February 1994). One sample from each pair was used in the analysis, the remaining sample being stored at 4 °C in case a check analysis was required.

Vehicle:

yes

Details on test solutions:

The test substance was dissolved in 1 % Tween 80 tetrahydrofuran to give an initial stock Solution of 10 mg/mL. Serial dilutions of this stock Solution were prepared with the auxiliary solvent and aliquots added to test water to give the desired series of exposure levels.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Straus
- Age of parental stock (mean and range, SD): Gravid adults were isolated 24 hours prior to Initiation of the test. Young daphnids produced overnight were used for testing.
- Feeding during test: Yes
- Food type: mixed unicellular algal culture
- Amount: 5 mL
- Frequency: daily

ACCLIMATION
- Acclimation conditions: same as test
- Type of food: Suspension of mixed algae (predominantly Scenedesmus and Selenastrum spp).
- Feeding frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

21 ± 1°C

pH:

7.7 - 7.8

Dissolved oxygen:

8.1 - 8.2 mg/L

Nominal and measured concentrations:

Nominal test concentrations: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
Measured test concentrations: 0.010, 0.029, 0.089, 0.28 and 0.83 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: flask
- Type: open
- Material, fill volume: glass, 400 mL
- Aeration: No
- Renewal rate of test solution: three times per week (Monday, Wednesday and Friday)
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 medium was prepared using analytical grade reagents and reverse osmosis purified water (see "Any other information on materials and methods").
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light : 8 hours dark

EFFECT PARAMETERS MEASURED
The live and dead Daphnia of the "parental" (P) generation were counted daily and the numbers recorded together with observations on the general condition and size of the Daphnia as compared with the controls. The numbers of live and dead "filial" (F) Daphnia, the numbers of "parental" (P) Daphnia with eggs or young in the brood pouch, and the numbers of discarded unhatched eggs were counted and recorded immediately prior to test media renewal (i.e. three times per week).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.125, 3.2

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.83 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (total fraction)

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

EC50

Basis for effect:

immobilisation

Remarks on result:

not determinable

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

>= 0.83 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat. (total fraction)

Basis for effect:

reproduction

Remarks on result:

other: Since the EC50 values are so far in excess of the water solubility value, a meaningful EC50 cannot be quoted.

Details on results:

No significant mortalities occurred in Daphnia magna exposed to a range of test item concentrations for 21 days. Consequently, it was not possible to calculate EC50 (immobilisation) values.
The "no effect" level (immobilisation <20 %) at 21 days was >0.83 mg/L. Background mortalities ranged from 20 – 22 % and it is thought, therefore, that the 27 % immobilisation obtained at the highest test concentration is not significantly different. Higher test concentrations could not be prepared owing to the limited solubility of the test item in water (0.01 mg/L) and having regard to the amount of auxiliary solvent permitted in die test. It should be noted, that a percentage of the mortalities observed in all but the highest exposure level (1.0 mg/L) and controls were probably due to what was thought to be a fungal infection.
The 14- and 21- day EC50 (reproduction) values have been estimated to be greater than 0.83 mg/L, Since the EC50 values are so far in excess of the water solubility value, meaningful EC50 cannot be quoted. There were no other apparent differences in size, activity or any other characteristics between Daphnia of the control and treatment groups.
Information on the effects of the test item on the generation is limited since, by study design, the young are removed soon after liberation from the brood pouch. Numbers of unhatched eggs and dead young were low in all control and treatment groups ( < 1 per female).
The "no-observed effect concentration" (21 days) has been estimated to be >0.83 mg/L.

Table 2: Validation Criteria

	Required	Actual
Surfactant control	≤ 20 %	20 %
Dissolved oxygen	≥ 60 % ASV	≥ 85 % ASV
pH (Surfactant control)	Deviation ≤ 0.3	0.5+
First young (Surfactant control group)	Produced within 9 days	9 days
Cumulative young per female (control group)	≥ 40	41
Number of broods per solvent control group	≥ 3	≥ 3*
* Pooling of data from the 4 replicates tends to mask the identification of distinct broods but at least 3 broods are considered to have been produced given the numbers of young counted and the length of the reproductive phase in relation to the typical breeding pattern of Daphnia magna + Although the Variation in pH is greater than 0.3 units, this was only observed from Days 0 to 2. Throughout the rest of the study, the deviation remained <0.3 and it is considered, therefore, that this in no way affects the validity of the results ASV Air Saturation Value

Table 3: Summary of the results

Nominal concentration mg/L	Measured concentration mg/L	% Survival of P1	No. live young		No. dead young		No. unhatched eggs
			Total	Per♀	Total	Per♀	Total	Per♀
Control	Control	78	1354	43	2	<1	2	<1
Surfactant control	Surfactant control	80	1391	41	5	<1	9	<1
0.010	0.010	68	1099	39	1	<1	3	<1
0.032	0.029	40	780	51	1	<1	2	<1
0.10	0.089	68	1322	49	0	0	8	<1
0.32	0.28	68	1287	47	1	<1	13	<1
1.0	0.83	73	1242	37	0	0	0	0

Validity criteria fulfilled:

yes

Conclusions:

In a long term toxicity study in Daphnia magna according to OECD guideline 202 (Part II), a NOEC of above 0.83 mg/L was determined (No EC50 could be derived).

Executive summary:

The long term aquatic toxicity to daphnia was examined according to OECD guideline 202, part II (Huntingdon, 1992). Water flea (daphnia magna) were exposed to the test substance at nominal concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L (measured: 0.010, 0.029, 0.089, 0.28 and 0.83 mg/L) under semi-static conditions. Continuous flow conditions could not be used due to the poor solubility of the test substance in water. Media was renewed at 48 - 72 hour intervals.

No significant mortalities occurred in Daphnia magna exposed to a range of test item concentrations for 21 days, Consequently, it was not possible to calculate EC50 (immobilisation) values.

The "no effect" level (immobilisation <20 %) at 21 days was >0.83 mg/L. Background mortalities ranged from 20 – 22 % and it is thought, therefore, that the 27 % immobilisation obtained at the highest test concentration is not significantly different. Higher test concentrations could not be prepared owing to the limited solubility of the test item in water (0.01 mg/L) and having regard to the amount of auxiliary solvent permitted in die test. It should be noted, that a percentage of the mortalities observed in all but the highest exposure level (1.0 mg/L) and controls were probably due to what was thought to be a fungal infection.

The 14- and 21- day EC50 (reproduction) values have been estimated to be greater than 0.83 mg/L, Since the  EC50 values are so far in excess of the water solubility value, meaningful EC50 cannot be quoted. There were no other apparent differences in size, activity or any other characteristics between Daphnia of the control and treatment groups.

Information on the effects of the test item on the generation is limited since, by study design, the young are removed soon after liberation from the brood pouch. Numbers of unhatched eggs and dead young were low in all control and treatment groups ( < 1 per female). The "no-observed effect concentration" (21 days) has been estimated to be >0.83 mg/L.

No adverse effects on survival or reproduction were observed in Daphnia magna exposed to the test item for 21 days. It should be noted that concentrations required to cause impairment of reproduction are unlikely to occur in the environment due to the low solubility of the test item in water (0.01 mg/L).

Description of key information

In the long-term toxicity key study with Daphnia magna a 21d-EC50 of > 0.53 mg/L based on reproduction was determined and an EC10 of 0.027 mg/L based on reproduction was calculated (Huntingdon, 1992). The NOEC based on immobilization was >= 0.53 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.027 mg/L

Additional information

Key study

The long term aquatic toxicity to daphnia was examined according to OECD guideline 202, part II (Huntingdon, 1992). Water flea (daphnia magna) were exposed to the test substance at nominal concentrations of 0.012, 0.045, 0.17 and 0.53 mg/L under semi-static conditions. Continuous flow conditions could not be used due to the poor solubility of the test substance in water. Media was renewed at 48 - 72 hour intervals.

There were no indications of acute or prolonged effects on the parental generation at concentrations well in excess of the water solubility value. Prolonged exposure of Daphnia magna to the test item resulted in a slight reduction in the numbers of young produced at the three highest exposure levels (0.045, 0.17 and 0.53 mg/L).

No other adverse effects on survival or reproduction were observed. It should be recognized that the concentrations required to cause impairment of reproduction are unlikely to occur in the environment due to the low solubility of the test item in water. The NOEC based on measured concentrations is above the water solubility value of the test substance.

The NOEC/EC10 for immobility was recorded as >0.53 mg/L based on the measured concentrations. The EC10 for reproduction was statistically calculated as 0.027 mg/L using the Williams test with a lowest effect concentration of 0.045 mg/L.

Supporting study

The long term aquatic toxicity to daphnia was examined according to OECD guideline 202, Part II (TNO, 1995). Water flea (Daphnia magna) were exposed to the test substance at concentrations above the water solubility under semi-static conditions. The chemical analytical results of the daphnia test suffered from a consistently higher detection limit. The calibration standards used in the analysis of the daphnia test samples (0.1 to 2 mg/L) laid outside (above) the range of the samples to be analyzed. The calibration solutions contained 0.5 to ca. 10 % acetonitrile, whereas the injected daphnia test samples contained no organic solvent. Given the hydrophobic nature of the test material, this method was not ideal. The potential discrepancy in the results which can be attributed to this is difficult to determine.

It is concluded that the test item is not toxic to Daphnia magna, with respect to reproduction and survival within its aqueous solubility, when tested as an aqueous extract, prepared by mechanical stirring. This is a result which is consistent with what may be expected on basis of the molecule size of the test item (M= 532). An EC50 and a NOEC value equal to or above the water solubility were determined.

In accordance with Article 25(3) of the REACH Regulation the robust study summaries submitted 12 years previously, are additionally used for the purpose of the registration.

Supporting study

A long term toxicity study according to OECD guideline 202, part II was performed (RCC Holding, ECHA RSS, 2017). Daphnia magna were exposed to a nominal concentration of 10 mg/L (mean measured concentration: 1.9 mg/L). This was the highest concentration attainable without observable separation of the test substance.

20 mg/L Tween 80 were used as auxiliary solvent for dispersal. No adverse effects or treatment-related mortalities observed up to the highest concentration tested. An EC50 of above 1.9 mg/L was determined. The NOEC was set to 1.9 mg/L exactly.