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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2021 - February 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
test item is unstable in water, therefore the hydrolysis product PMA was tested
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Storage Conditions: Store in cool place. Keep container tightly closed in a dry and well-ventilated place
Analytical monitoring:
yes
Details on sampling:
Duplicate samples (10mL) were taken from the freshly prepared test medium of all test concentrations and from the control at days 0, 2, 5, 12 and 16. At the start of the renewal period, the samples were taken before the test media were divided into replicate vessels.
Stability samples were taken at the end of the test medium renewal periods of 48 hours (days 2, 7 and 14) and at the end of two renewal periods of 72 hours (days 5 and 19). For the 48- and 72-hour stability samples, the contents of the respective replicates were combined prior to sampling.
The concentrations of the test item were measured in at least one of the duplicate from the control and the nominal test item concentrations of 32 and 100 mg/L from all sampling dates. Due to a drop in measured concentrations at the highest nominal concentration group of 100 mg/L, the analysis was repeated and the second of the duplicate samples (retain sample) was analyzed as well.
The samples from the nominal test concentrations of 1.0 to 10 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test.

- Sample storage conditions before analysis: All individual samples were frozen (at -20 ± 5 °C) immediately after sampling and stored until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Prior to the start of the test and prior to each test medium renewal, the highest nominal concentration of 100 mg/L was freshly prepared by completely dissolving approx. 150 mg of the test item in 1500 mL of test water (Elendt M7 medium) using ultrasonic treatment for 15 minutes and intense stirring for 15 minutes at room temperature. After the stirring period the pH was adjusted from pH 4.1-4.3 to pH 6.7-6.8 with 1 M sodium hydroxide solution. The adjusted test media of the highest test concentration were diluted with test water to obtain the test media of the additional test item concentrations
- Controls: test water only
- Test concentration separation factor: approx 3.2
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
The study was performed with females of the species Daphnia magna Straus. A clone of this species (originally from the Daphnia Collection of the University of Basel/Switzerland in 2015) is successfully bred in IES Ltd Laboratories in culture medium identical to the medium used for the test. The temperature and light conditions were identical to those of the tests.
During breeding, daphnids were generally fed three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen/Germany) and cultivated at IES Ltd Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA GmbH, 49324 Melle/Germany).
Each stock animal was maintained separately in a 100 mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.
The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old, by removing the offspring <24 hours before start of the test.
The test method and test species, Daphnia magna, are recommended by the test guideline
The test animals were fed daily with a food mixture containing a suspension of green algae of the species Desmodesmus subspicatus (freshly grown at IES Ltd) and a fish food suspension. The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (Tetra Min Hauptfutter, obtained from TETRA GmbH, 49324 Melle/Germany) in 500 mL of test medium. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test medium and boiled. The suspension was stored deep-frozen in small quantities until use.
The carbon contents of the algal and fish food suspensions were determined using a vario TOC cube from Elementar Analysensysteme GmbH, Langenselbold, Germany. The food amount (in a ratio of 1:1, based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
Water Hardness: 2.5 mmol/L (= 250 mg/L as CaCO3)
Alkalinity: 0.9 mmol/L
Test temperature:
The water temperature was maintained between 20 and 21 °C during the test period.
pH:
During the test period, the pH of the test medium and control ranged from 6.7 to 8.3
Dissolved oxygen:
The dissolved oxygen concentrations were at least 8.0 mg/L
Salinity:
Freshwater used
Nominal and measured concentrations:
Nominal: 0, 1, 3.2, 10, 32, 100 mg/L
Mean measured: 32, 79 mg/L (only the top two concentrations were measured as the other doses were below the NOEC).

Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume: 100 mL, filled with 80 mL of test solution. Covered with a glass loate to reduce loss of water and to avoid the entry of dust into the solutions.
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): every 48 or 72 hours (Monday, Wednesday and Friday).
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The test water (Elendt M7 medium, per OECD TG 211) was prepared by dissolving the required analytical grade salts and additives in ultrapure water.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: A 16-hour light to 8-hour dark cycle with a 30-minute transition period was used.
- Light intensity: Light intensity during the light period was between 17 and 18 µmol m-2 s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobility of adults or other visible abnormalities - daily
Number of live offspring - daily
Body length of adults - day 21

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx 3.2
- Range finding study
- Test concentrations: 0, 1.0, 10, 100 mg/L nominal
- Results used to determine the conditions for the definitive study:

Nominal No. of Number of Living % Inhibition
concn Daphnids offspring after [%]
[mg/L] 12 Days
Control 10 524 -
1.0 10 498 5
10 10 468 11
100 10 442 16

Reference substance (positive control):
yes
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
growth
Details on results:
- Behavioural abnormalities: none reported
- Observations on body length and weight: The mean body length of the daphnids in the control was 4.41 ± 0.13 mm (Table 4 below and Figure 2 attached). No significant inhibitory effect of the test item on the body length was determined in any treatment concentrations. Therefore, the NOEC and LOEC for body length was determined to be 79 and >79 mg/L, respectively (Dunnett’s Multiple t-test Procedure, α=0.05, one-sided smaller). The EC10 and EC50 value for body length could not be calculated because none of the responses exceeded 10 %. Therefore, the EC10 and EC50 for body length were determined to be >79 mg/L.
- Other biological observations:
- Mortality of control: no control animals died during the study
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
No positive control
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional®.
For the determination of the 21-day NOEC, the mean reproduction rates and the mean body lengths of the Daphnia or daphnids at the test concentrations were compared to the control by Dunnett’s Multiple t-test procedure (one-sided smaller,  = 0.05). The mortality of the Daphnia at the test concentrations were compared to the control by Fisher`s Exact Binomial Test with Bonferroni Correction (one-sided greater,  = 0.05).

Analytical results

The analytically determined concentrations of the test item in the test media of the nominal concentrations of 32 mg/L were between 96 and 102 % of the nominal values during the test period. Four out of five freshly prepared samples of the concentration 100 mg/L showed values ranging from 97 to 99 %. The freshly prepared sample on day 16 showed a lower recovery of 63 %, which was confirmed by repeat analysis and analysis of the retain sample. However, it can be assumed that the solution was prepared correctly, since the lower concentration of 32 mg/L with a recovery of 99 % was diluted from this concentration. It is suspected that an error was made during sampling.The recoveries of the aged samples from the concentration 100 mg/L ranged between 64 to 75 %. The losses of the test item in these samples might be due to some solubility limitations at this concentration in connection with the content of nutrient salts and algae food in these samples. Therefore, the biological results were based on mean measured concentrations, calculated as time-weighted means of the test item concentrations measured at the start of the test medium renewals and the concentrations measured in the samples at the end of the renewal periods.

 

Nominal Test Item Concentration [mg/L]

32

100

 

Measured Test Item Concentrations [mg/L]

Sampling Day / Sample Age

0/0 (fresh)

30.8

98.2

2/48 (aged)

32.4

68.5*

2/0 (fresh)

31.5

97.5

5/72 (aged)

32.8

64.5*

5/0 (fresh)

32.1

97.2

7/48 (aged)

32.7

75.4*

12/0 (fresh)

32.0

97.2

14/48 (aged)

31.3

73.5*

16/0 (fresh)

31.6

62.7*

19/72 (aged)

31.7

60.5*

Mean Measured Concentration [mg/L]

32

79

*: mean of three measurements

The samples from the nominal test concentrations of 1.0 to 10 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

Survival

The mortality of the parental daphnids is shown in Table 1.

In the control and the highest nominal concentration of 100 mg/L, all test animals survived. At nominal concentration of 1.0 mg/L, 80% of the test animals survived and at nominal concentrations of 3.2, 10, and 32 mg/L, 90% of the daphnids survived until the end of the test. These mortalities were regarded as natural and not considered to be due to a toxic effect of the test item. According to the test guideline a mortality of up to 20 % in the control is accepted for the test to be valid. Moreover, neither the reproduction nor the length of the daphnids was significantly reduced at these concentrations and not part of the dose-response curve, as no mortality was observed at the highest nominal concentration of 100 mg/L.

The NOEC and LOEC for mortality were determined to be at the mean measured concentration of 79 mg/L and>79 mg/L, respectively (Fisher`s Exact Binomial Test with Bonferroni Correction,α=0.05, one-sided greater).

The EC10 and EC50 for mortality were determined to be > 79 mg/L.

Table 1 Number of Surviving Test Animals

Exposure Time
[Days]

Number of Surviving Test Animals

Nominal Test Item Concentration

(Mean Measured Test Item Concentration)

[mg/L]

Control

1.0
(n.a.)

3.2
(n.a.)

10
(n.a.)

32
(32)

100
(79)

0

10

10

10

10

10

10

1

10

10

10

10

10

10

2

10

10

10

10

10

10

3

10

10

10

10

10

10

4

10

10

10

10

10

10

5

10

10

10

10

10

10

6

10

10

10

10

10

10

7

10

10

10

10

10

10

8

10

10

10

10

9

10

9

10

10

10

10

9

10

10

10

10

10

9

9

10

11

10

10

10

9

9

10

12

10

10

10

9

9

10

13

10

10

10

9

9

10

14

10

10

10

9

9

10

15

10

9

10

9

9

10

16

10

9

9

9

9

10

17

10

9

9

9

9

10

18

10

9

9

9

9

10

19

10

8

9

9

9

10

20

10

8

9

9

9

10

21

10

8

9

9

9

10

% Surviving on Day 21

100

80#

90#

90#

90#

100

 #: The mortality of 10-20 % was regarded as natural and not as a toxic effect of the test item.

n.a.: Not analyzed, since below the NOEC.

Reproduction rate

The first young offspring released from their parent animals were recorded in the control and all test concentrations at observation on Day 8. The cumulative number of offspring and the mean reproduction rates of the daphnids are shown in Table 2 and Table 3 below and Figure 1 attached.  

The mean reproduction rate of the daphnids in the control was 160 ± 28 living offspring per surviving adult (mean ± standard deviation).

No significant inhibitory effect of the test item on the mean reproduction rate was determined in any treatment concentration.

The NOEC for mean reproduction rate was determined to be the mean measured test item concentration of 79mg/L (Dunnett’s Multiple t-test Procedure,α=0.05, one-sided smaller). The LOEC was determined to be >79 mg/L.

The EC10 and EC50 value for reproduction rate could not be calculated because none of the responses exceeded 10 %. Therefore, the EC10 and EC50 for reproduction rate were determined to be >79mg/L.

Table 2: Total Number of Living Young Daphnids Produced until the End of the Test (Cumulative Values)

Exposure Time
[Days]

Number of Living Young Daphnids

Nominal Test Item Concentration

(Mean Measured Test Item Concentration)

[mg/L]

Control

1.0
(n.a.)

3.2
(n.a.)

10
(n.a.)

32
(32)

100
(79)

0

0

0

0

0

0

0

1

0

0

0

0

0

0

2

0

0

0

0

0

0

3

0

0

0

0

0

0

4

0

0

0

0

0

0

5

0

0

0

0

0

0

6

0

0

0

0

0

0

7

0

0

0

0

0

0

8

97

116

81

97

96

37

9

104

116

109

97

96

45

10

104

116

109

97

96

45

11

355

367

348

330

273

271

12

355

367

348

330

273

271

13

355

367

348

330

273

271

14

658

695

668

647

575

573

15

658

695

668

647

575

573

16

690

695

710

647

675

673

17

1045

1046

1029

985

1004

977

18

1130

1046

1076

1012

1004

1046

19

1130

1046

1076

1012

1004

1046

20

1561

1390

1308

1326

1351

1302

21

1603

1425

1458

1483

1351

1450

Number of Adults
at Test End

10

8#

9#

9#

9#

10

 #:  The mortality of 10-20 % was regarded as natural and not as a toxic effect of the test item.

n.a.: Not analyzed, since below the NOEC.


 Table 3     Number of Living Offspring Produced per Adult after 21 Days of Exposure

Replicates

Number of Living Offspring

Nominal Test Item Concentration

(Mean Measured Test Item Concentration)

[mg/L]

Control

1.0
(n.a.)

3.2
(n.a.)

10
(n.a.)

32
(32)

100
(79)

1

137

125

150

149

(0)#

130

2

188

152

170

182

181

132

3

168

192

(59)#

168

134

136

4

171

197

171

192

142

153

5

116

186

152

173

143

127

6

140

156

143

143

161

151

7

167

(77)#

163

132

161

151

8

129

114

84

(10)#

166

155

9

194

(66)#

190

178

155

151

10

193

160

176

156

108

164

Mean

160

160

155

164

150

145

± SD

28

30

30

20

21

13

n

10

8

9

9

9

10

CV %

18

19

20

12

14

8.7

% of Control

100

100

97

102

94

91

STAT

-

n.s.

n.s.

n.s.

n.s.

n.s.

SD: Standard Deviation

CV %: Coefficient of variation: (SD/mean) × 100 %

#: The adult daphnia died during the exposure. In parentheses the number of offspring from this daphnia. The mortality of 20 % was regarded as natural. The calculation of the mean reproduction rate is therefore based on the surviving adults only (according to the OECD Guideline No. 211, 2012).

n.s.: Statistically not significantly different compared to thecontrol (Dunnett’s t-test, one-sidedsmaller, α= 0.05)

n.a.: Not analyzed, since below the NOEC.

 

Body length

Table 4 Body Length [mm] of the Surviving Adults after 21 Days of Exposure

Replicates

Body Length [mm]

Nominal Test Item Concentration

(Mean Measured Test Item Concentration)

[mg/L]

Control

1.0
(n.a.)

3.2
(n.a.)

10
(n.a.)

32
(32)

100
(79)

1

4.53

4.27

4.27

4.47

 -

4.33

2

4.40

4.40

4.47

4.53

4.40

4.33

3

4.47

4.47

 -

4.47

4.27

4.33

4

4.47

4.47

4.55

4.40

4.40

4.33

5

4.07

4.40

4.40

4.47

4.47

4.27

6

4.40

4.47

4.47

4.47

4.47

4.40

7

4.47

 -

4.47

4.40

4.40

4.20

8

4.40

4.40

4.40

 -

4.33

4.40

9

4.47

 -

4.33

4.47

4.40

4.33

10

4.47

4.47

4.47

4.53

4.47

4.47

Mean

4.41

4.42

4.42

4.47

4.40

4.34

± SD

0.13

0.069

0.085

0.047

0.067

0.073

n

10

8

9

9

9

10

CV %

2.9

1.6

1.9

1.1

1.5

1.7

% of Control

-

100

100

101

100

98

STAT

-

n.s.

n.s.

n.s.

n.s.

n.s.

 SD: Standard Deviation

CV %: Coefficient of variation: (SD/mean) × 100 %

-: No measurements as the parental daphnid did not survive until the end of the test

n.s.: Statistically not significantly different compared to the control (Dunnett`s t-test, one-sided smaller, α= 0.05)

n.a.: Not analyzed, since below the NOEC.

 

Validity of the test

The test was considered to be valid since all relevant validity criteria have been fulfilled according to the OECD Guideline No. 211 (2012).

 -      Mortality of parent test animals in the control should not exceed 20 % at the end of the test and was 0 % in the actual test.

 -      The mean number of living offspring in the control should be at least 60 per surviving adult Daphnia at the end of the test and was 160 in the actual test.

 -      The coefficient of variation around the mean number of living offspring produced per parent animal in the control should be <= 25 % and was 17.5 % in the control in the actual test.

 

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, taking into account the effects of the test item on survival, growth, and reproduction of the test animals, the overall 21 day NOEC was determined as 79 mg/L (mean measured), since no toxic effects were observed on the Daphnia magna at this and all lower test concentrations.
Executive summary:

As PMDA is unstable in water, the corresponding hydrolysis product Pyromellitic acid (PMA) was tested.

The impact of the test item on the survival, growth (body length),andreproduction rate ofDaphnia magnawas investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2012): “Daphnia magnaReproduction Test”.

The nominal test item concentrations tested were 1.0, 3.2, 10, 32, and 100 mg/L. Additionally, a control group was tested in parallel.

The analytically determined concentrations of the test item in the test media of the nominal concentrations of 32 mg/L were between 96 and 102 % of the nominal values during the test period.Four out of five freshly prepared samplesof the concentration 100 mg/Lshowed values ranging from 97 to 99 %.The freshly prepared sample on day 16 showed a lower recovery of 63 %, which was confirmed by repeat analysis and analysis of the retain sample. However, it can be assumed that the solution was prepared correctly, since the lower concentration of 32 mg/L with a recovery of 99 % was diluted from this concentration. It is suspected that an error was made during sampling.The recoveries of the aged samples fromthe concentration 100 mg/L ranged between 64 to 75 %.The losses of the test item in these samples might be due to some solubility limitations at this concentration in connection with the content of nutrient salts and algae food in these samples.

Therefore, the biological results were based on mean measured concentrations, calculated as time-weighted means of the test item concentrations measured at the start of the test medium renewals and the concentrations measured in the samples at the end of the renewal periods.

The samples from the nominal test concentrations of 1.0 to 10 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

The impact of the test item on thesurvival, growth (body length),andreproduction rate ofDaphnia magnacan be summarized as follows (based on mean concentrations):

 

Biological Endpoints

Nominal Test Item Concentration
(Mean Measured Test Item Concentration)
[mg/L]

 

Control

1.0
(n.a.)

3.2
(n.a.)

10
(n.a.)

32
(32)

100
(79)

Survival [%] after 21 days of exposure

100

80#

90#

90#

90#

100

Mean reproduction rate

160

160

155

163

150

145

Mean reproduction rate in % of control

--

100

97

102

94

91

Mean body length of the adults [mm]

4.41

4.42

4.42

4.47

4.40

4.34

Mean body length in % of control

--

100

100

101

100

98

 #: The mortality of 10 and 20 % in the nominal concentrations of 1.0 to 32 mg/L was regarded as natural and not considered to be due to a toxic effect of the test item as according to the test guideline a mortality of up to 20 % in the control is accepted for the test to be valid. Moreover, neither the reproduction nor the length of the daphnids were significantly reduced at these concentrations and no mortality was observed in the highest concentration. The calculation of the mean reproduction rate is therefore based on the nine or eight surviving adults only (according to the OECD Guideline No. 211, 2012).

n.a.: not analyzed since below the NOEC

In conclusion, taking into account the effects ofthe test itemon survival, growth, and reproduction of the test animals, the overall 21 day NOEC was determined as 79 mg/L (mean measured), since no toxic effects were observed on theDaphnia magnaat this and all lower test concentrations.

Description of key information

As PMDA is unstable in water, the corresponding hydrolysis product Pyromellitic acid (PMA) was tested.

The impact of the test item on the survival, growth (body length), and reproduction rate of Daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2012): “Daphnia magnaReproduction Test”. The nominal test item concentrations tested were 1.0, 3.2, 10, 32, and 100 mg/L. Additionally, a control group was tested in parallel. The biological results were based on mean measured concentrations, calculated as time-weighted means of the test item concentrations measured at the start of the test medium renewals and the concentrations measured in the samples at the end of the renewal periods.The samples from the nominal test concentrations of 1.0 to 10 mg/L were not analyzed, since these concentrations were below the NOEC determined in this test and, thus, were not relevant for the interpretation of the biological results.

Taking into account the effects of the test item on survival, growth, and reproduction of the test animals, the overall 21-day NOEC was determined as 79 mg/L (mean measured), since no toxic effects were observed on the Daphnia magna at this and all lower test concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
79 mg/L

Additional information