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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jun. 12,2016 to Jun.26,2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
Guideline for the testing of chemicals 203. Fish, Acute Toxicity Test. OECD, 1992.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
EC Directive 2001/59, Part C.1 Acute Toxicity for Fish. 0.J. L142, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Version / remarks:
Ecological Effects Test Guidelines, OPPTS 850.1075. Fish Acute Toxicity Test, Freshwater and Marine. EPA 712-C-96-118, 1996.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
No further details specified in the study report.
Analytical monitoring:
yes
Details on sampling:
100 mL water samples were taken (at least in duplicate) from each concentration during the definitive test at 0, 24, 48, 72 and 96 h. On each occasion, one sample was analysed after certain pre-treatments; the remaining samples were retained in case further analysis would be required.
Vehicle:
no
Details on test solutions:
In the range-finding test, the test solution was prepared by directly adding appropriate amounts of MLA-3202 in dilution water and then facilitating its dispersion by stirring for 30 min. The 0.10 mg/Land 1.00 mg/L test solutions were colourless and clear. The 10.0, I 00 mg/L test solutions were insufficiently soluble and turbid.
In the definitive test, the test medium was prepared as a slow stir stock solution. The test solution was prepared by adding 1.0035 g MLA-3202 in 10 L dilution water. The aqueous test substance mixture was stirred for 1 hrs on a magnetic stirplate and a telfon stirbar at 3 5°C. The vortex height was set at least 10% of the liquid height. At the end of the 1 h, stirring was stopped. The stock solution stood for 2 hour at room temperature prior to the removal of any undissolved test item by filtration through 0.45 μm millipore membrane to produce the stock solution of the test item. The test solutions were diluted by the stock solution.
Test organisms (species):
other: Rare minnow, Gobiocypris rarus
Details on test organisms:
The test species Rare minnow, Gobiocypris rarus (Batch No. F20160415G), were obtained from a fish supplier ofl nstitute of Hydro biology, Chinese Academy of Sciences.
Fish were held at least for 12 days in holding tanks supplied with a continuous flow of aerated water before being used for testing. Fish to be used in the test were held for 7 days in water of the quality and temperature to be used in the test.
A photoperiod of 16 hours light, provided by overhead fluorescent tubes, and 8 hours dark was maintained. The oxygen concentration was more than 60% of the air saturation value.
The fish was fed daily during the holding period on proprietary fish food. They were held without food for approximately 24 hours before being placed into the test vessels. The ingredients of the fish food are given as follows:
Crude Protein> 36.0%
Crude Fat> 2.0%
Crude Fibre< 3.0%
Crude Ash< 13.5%
Moisture< 10.0%
Characteristics of the fish food are measured at least twice a year by Jiangsu Provincial Center for Disease Prevention and Control.
During the holding period the tanks was inspected daily and any debris or unhealthy or dead fish removed.
After the 48 hour settling-in period, no mortality was observed in the following 7 days. So the batch offish (Batch No.: F20160415G) was accepted.
The average wet-weight and length of the fish used to the test was 0.222 g and 2.75 cm, and the relative standard deviation (RSD) was 7.75% and 4.61 %, respectively.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
As per guideline
Post exposure observation period:
No post exposure observation period specified
Hardness:
164 mg (CaC03)/L to 175 mg (CaC03)/L.
Test temperature:
22.8°C to 23.1°C
pH:
7.72 and 7.97
Dissolved oxygen:
75% ~ 97% of the air saturation
Salinity:
Not applicable - freshwater study
Conductivity:
Not specified
Nominal and measured concentrations:
Nominal concentrations of 0.10, 1.00, 10.0 and 100 mg/L were used in the range-finding test.
Nominal concentrations of 1 %, 2%, 4%, 6%, 8% and 10% stock solution were used in the definitive test.
Details on test conditions:
Observations and Evaluations
During the test, all kinds of abnormal responses of the fish observed were recorded, such as mortality, inactivity, abnormal swimming pattern, other abnormal behaviour, etc. Fish were considered dead if there was no visible movement (e.g. gill movements) and if touching of the caudal peduncle produced no reaction.

Range-finding Test
The range-finding test, carried out under static conditions, was conducted to determine the range of concentrations for the subsequent test.
In the range-finding test, groups of fish (5 per group) were exposed to the test solutions with nominal concentration of 0.10, 1.00, 10.0 and 100 mg/L. One control group was also included in the study using test water without the test substance. For each test tank 3 L test solution was filled in. No replicates were used.
The test fish were randomly chosen and put into appropriate test solutions after the temperature had been adjusted to the required value. This was done in 30 minutes.
During the test, the following conditions were maintained:
-Light: 16 hours photoperiod daily (light intensity: 1000 to 1500 lux);
-Temperature: 23.0°C to 23.5°C;
-Oxygen concentration: 80% - 95% of the air saturation; No aeration.
-Feeding: none.
The test duration was 96 hours. Dead fish was removed at least once daily and discarded.
The mortalities of the fish were recorded at 24, 48, 72 and 96 h, and then the maximum concentration causing no mortality (96 h-LC0) and the minimum concentration causing 100 % mortality (LC100) were determined.

Definitive Test
A static method was adopted in definitive test. The stability of the test solution is confirmed by results indicated in Table 4 (deviation within 20%). Based on the results of the range-finding test, the concentration of 1 %, 2%, 4%, 6%, 8% and 10% stock solution was assigned in definitive test. Synchronously a blank control was used in the test. No replicate was assigned for each treatment group and control group, while the initial number of testing fish was 10 for each group.
The test fish were randomly chosen and put in different test solutions after the temperature has been adjusted to the required value. This was done in 30 minutes.
During the test, the following conditions were maintained:
-Light: 16 hours photoperiod daily (light intensity: 1000 lux to 1500 lux);
-Temperature: 22.8° C to 23. 1° C;
-Oxygen concentration: 75% - 97% of the air saturation; No aeration.
-Feeding: none.
At 3, 6, 24, 48, 72 and 96 h, the mortalities of the fish were recorded, and observations on individual behaviour were performed. Meanwhile, measurements of pH, dissolved oxygen and temperature were carried out and recorded daily.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate,
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.24 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
0.858 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
Analytical Method for Determination of the test substance in Water
Specificity
Under the GC-FID condition, at the retention time of 3.1 min, 3.4min, 3.6min, 4.5min and 5.1 min, the chromatographic peak emerged for the test substance sample and there was no chromatographic peak emerged for the blank sample (Figure 4). So the GC-FID method was specific for MLA-3202.

Calibration curve
A series of standard solutions with concentration at 0.00, 5.76, 11.5, 23.0, 46.1, 92.2 and 115 mg/L were measured under the GC-FID conditions mentioned above. Concentrations of the test substance were quantified by GC-FID using two of the principal peaks which were considered representative of the different physico-chemical characteristics of the individual components making up the complex nature. Based on the test result, a linear regression equation was obtained between the concentration and the GC-FID response:
A=3864.7c+454.47, with good linearity of r2 = 0.9975, where A represents peak area (μV*s); and c is the concentration of the test substance (mg/L). The results show that linearity for concentration range of 0.00 mg/L to 115 mg/L is good.

Precision
Under the above condition, 115 mg/L standard solutions of the test substance were analysed for 6 times. The relative standard deviations were 2.97%.

Recovery Test
The recovery samples with concentrations of 0.115 mg/L were prepared by adding 0.10 mL standard solution II (115 mg/L) to a total volume of 100 mL test water with three replications. 100 mL recovery samples were extracted with 50 mL dichloromethane for 15 min and repeated once, combined twice organic phase. The organic phase was concentrated by rotary evaporation to near dryness. The concentrate was diluted to total volume to 1.00 mL with n-hexane (concentrated 100 times), then the dilution was analysed by GC-FID.
The recovery rate was 80.9% ~ 88.7% for the concentration of 0.115 mg/L. The relative standard deviation was 4.66%.
The recovery samples with concentrations of 1.15 mg/L were prepared by adding 0.10 mL standard solution I (1152 mg/L) to a total volume of 100 mL test water with three replications. 100 mL recovery samples were extracted with 50 mL dichloromelhane for 15 min and repeated once, combined twice organic phase. The organic phase was concentrated by rotary evaporation to near dryness. The concentrate was diluted to total volume to 1.00 mL with n-hexane (concentrated 100 times), then the dilution was analysed by GC-FID. The recovery rate was 88.7% ~ 94.8% for the concentration of 1.15 mg/L. The relative standard deviation was 3.34%.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
If the calculation is based on S/N~3, the LOD is 2.00 mg/L. If the calculation is based on S/N~lO, the LOQ is 5.00 mg/L.
In this analytical method, the minimum detection concentration for water sample is 0.05 mg/L.

Analysis of the test substance in Test Solutions
The measured concentrations of 1 %, 2%, 4%, 6%, 8% and 10% stock solution were 0.121, 0.240, 0.503, 0.691, 0.858 and 1.27 mg/L, respectively. The results indicated that concentration of test substance was stable (deviation within 20%) in the water during the test period. Thus static method used in the definitive test was reasonable.

Test Condition
During the whole test period, the pH values of the control mediums and lest mediums were between 7.72 and 7.97, the Dissolved Oxygen (DO) values varied from 75% M 97% of the air saturation, the temperature of the test meciimns w::is maintained in the range of 22.8°C to 23.1°C, and the tot::il h::irciness was in the range of 164 to 175 mg (CaC03)/L.

Mortality and Effects
During the test period, all fish in the control and treatment of 1% stock solution (measured concentration 0.1 21 mg/L) and 2% stock solution (measured concentration 0.240 mg/L) were alive and appeared normal. Effects (Fish lying on side or back) occurred at concentrations of 4% stock solution (measured concentration 0.503 mg/L) and above. All Fish were dead after 96h-exposure at concentration of 8% stock solution (measured concentration 0.858 mg/L) and 10% stock solution (measured concentration 1.27 mg/L).
Results with reference substance (positive control):
24h-LC50 in the range of 200 mg/L to 400 mg/L.
Sublethal observations / clinical signs:

Individual and Mean Fish Weights and Lengths

No. of the Subsample Fish

Length (cm)

Weight (g)

1

2.92

0.236

2

2.87

0.203

3

2.64

0.247

4

2.52

0.214

5

2.71

0.197

6

2.66

0.233

7

2.83

0.219

8

2.89

0.207

9

2.74

0.243

10

2.73

0.224

Mean

2.75

0.222

RSD (%)

4.61

7.75

 

Water Quality Parameters of Test Solutions during the Definitive Test

Nominal Concentration

Duration (h)

pH

Temperature (°C)

Dissolved oxygen (%)

Hardness (mg/L CaCO3)

0 (Blank control)

0

7.72

23.1

93

171

24

7.77

23.0

89

170

48

7.75

22.8

85

167

72

7.79

22.9

81

173

96

7.85

23.1

77

165

1% stock solution

0

7.79

23.1

95

173

24

7.80

23.0

90

166

48

7.78

22.8

87

169

72

7.83

22.9

83

175

96

7.91

23.1

79

165

2% stock solution

0

7.81

23.1

96

168

24

7.84

23.0

92

164

48

7.85

22.8

87

169

72

7.89

22.9

82

174

96

7.90

23.1

76

172

4% stock solution

0

7.80

23.1

94

166

24

7.83

23.0

88

172

48

7.87

22.8

83

171

72

7.94

22.9

80

175

96

7.94

23.1

75

168

6% stock solution

0

7.84

23.1

97

169

24

7.87

23.0

91

171

48

7.90

22.8

85

168

72

7.96

22.9

80

169

96

7.97

23.1

76

167

8% stock solution

0

7.85

23.1

95

169

24

7.92

23.0

90

173

48

7.90

22.8

86

168

72

7.91

22.9

82

172

96

NA

NA

NA

NA

10% stock solution

0

7.87

23.1

96

167

24

7.90

23.0

92

168

48

NA

NA

NA

NA

72

NA

NA

NA

NA

96

NA

NA

NA

NA

NA: not detected due to all test fish death on the day before

 

Toxicity of Potassium Dichromate toGobiocypris rarus

Nominal Concentration (mg/L)

Initial Number of Fish

The Number of the Dead Fish

6 h

12 h

18 h

24 h

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

100

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

200

10

0

0

1

2

10

0

0

1

2

10

0

0

1

2

300

10

0

1

2

3

10

0

1

2

3

10

0

1

2

3

400

10

2

3

5

6

10

2

3

5

6

10

2

3

5

6

500

10

4

5

8

10

10

4

5

8

10

10

4

5

8

10

LC50 (mg/L)

-

500

393

309

95% confidence limit (mg/L)

-

-

337~459

256~374

 

Mortality during the Range-finding test

Nominal concentration (mg/L)

Initial Number of Fish

The Number of the Dead Fish

24 h

48 h

72 h

96 h

0 (Blank control)

5

0

0

0

0

0.10

5

0

0

0

0

1.00

5

3

4

5

5

10.0

5

5

5

5

5

100

5

5

5

5

5

 

Mortality during the Definitive Test

Nominal Concentration

Measured Concentration (mg/L)1

Initial Number of Fish

The Number of the Dead Fish

3 h

6 h

24 h

48 h

72 h

96 h

0 (Blank Control)

ND2

10

0

0

0

0

0

0

1% stock solution

0.121

10

0

0

0

0

0

0

2% stock solution

0.240

10

0

0

0

0

0

0

4% stock solution

0.503

10

0

0

0

1

3

4

6% stock solution

0.691

10

0

0

1

3

6

8

8% stock solution

0.858

10

0

3

6

8

10

10

10% stock solution

1.27

10

4

7

10

10

10

10

LC50 (mg/L, based on the measured concentration)

0.850

0.720

0.560

0.500

95% confidence limit (mg/L, based on the measured concentration)

0.760-0.940

0.620-0.830

0.470-0.670

0.420-0.600

1The average of the measured concentrations at 0, 24, 48, 72, 96 h.2ND: not detected

 

Visual Observations during the Definitive Test

Nominal Concentration

Measured Concentration (mg/L)

Visual Observations

3 h

6 h

24 h

48 h

72 h

96 h

0 (Blank Control)

ND2

10NB

10NB

10NB

10NB

10NB

10NB

1% stock solution

0.121

10NB

10NB

10NB

10NB

10NB

10NB

2% stock solution

0.240

10NB

10NB

10NB

10NB

10NB

10NB

4% stock solution

0.503

10NB

10NB

8NB & 2SR

7NB & 2SR & 1dead

4NB & 3SR & 3dead

4NB & 2SR & 4dead

6% stock solution

0.691

10NB

9NB & 1SR

8NB & 1SR & 1dead

5NB & 2SR & 3dead

2NB & 2SR & 6dead

2SR & 8dead

8% stock solution

0.858

7NB & 3SR

5NB & 2SR & 3dead

1NB & 3SR & 6dead

2SR & 8dead

10 dead

10 dead

10% stock solution

1.27

4NB & 2SR & 4dead

3SR & 7dead

10 dead

10 dead

10 dead

10 dead

Note: NB – Normal behaviors; SR – Fish lying on side or back

Validity criteria fulfilled:
yes
Conclusions:
The results showed that under valid static test conditions, the 96 h-LC50 of the test substance to fish (Rare minnow, Gobiocypris rarus) was 0.500 mg/L, with 95% confidence limit of 0.420 mg/L ~ 0.600 mg/L (based on the measured concentration). The maximum tested concentration causing no mortality (96 h-LC0) was 0.240 mg/L. The minimum concentration causing 100% mortality (96 h-LC100) was 0.858 mg/L, i.e.:
96 h-LC50 = 0.500 mg/L, 95%CI 0.420 ~ 0.600 mg/L (based on measured concentration);
96 h-LC0 = 0.240 mg/L (measured concentration);
96 h-LC100 = 0.858 mg/L (measured concentration).
Executive summary:

Under static conditions, the acute toxicity of test substance (MLA-3202) to Rare minnow (Gobiocypris rarus) was conducted according to: "The guidelines for the testing of chemicals" (HJ/T 153-2004), "The Guidelines for the Testing of Chemicals, Effects on Biotic Systems" (the 2nd edition) (Beijing: China Environment Press. 2013), and with reference to Procedure 203 of the "Guidelines for Testing of Chemicals" of the OECD: "Fish, Acute Toxicity Test" (1992).

A range-finding test and then a definitive test were performed respectively. Nominal concentrations of 0.10, 1.00, 10.0 and 100 mg/L were used in the range-finding test. Nominal concentrations of 1 %, 2%, 4%, 6%, 8% and 10% stock solution were used in the definitive test. Water samples taken from the blank control and the treatments in the definitive test were analysed. Concentrations of the test substance were quantified by GC-FID using two of the principal peaks which were considered representative of the different physico-chemical characteristics of the individual components making up the complex nature. Concentrations of the test substance were quantified using GC-FID of the measured concentrations were 0.121, 0.240, 0.503, 0.691, 0.858 and 1.27 mg/L, respectively. The analytical results showed that the concentration of the test substance was consistent in the test medium throughout the 96-hour test period (deviation within 20%). Thus, a static procedure was reasonable.

In the range-finding test, 5 fish per treatment with no replicates were used. And 10 fish per treatment with no replicates were used in the definitive test. The fish were exposed for 96 hours to the test solution.

During the test period, the pH values of the control mediums and test mediums were between 7.72 and 7.97, the Dissolved Oxygen (DO) values varied from 75% ~ 97% of the air saturation at the test temperature, the temperature of the test mediums were maintained in the range of 22.8°C to 23.1°C, and the total hardness was in the range of 164 mg (CaC03)/L to 175 mg (CaC03)/L. All fishes in the control group were normal. With the same conditions, K1Cr201 was used as the positive control substance, and the resulting 24h-LC50 was 309 mg/L. So the study met the acceptability criteria prescribed by the protocol (The mortality of control :S 10%; pH: 6.0 ~ 8.5; dissolved oxygen concentration: >60% of the air saturation value; total hardness: 10 ~ 250 mg (CaC03)/L; temperature: (23±2)°C; 24h-LC50 of K1Cr201 in the range of 200 mg/L to 400 mg/L). Therefore, the test was considered valid.

During the test period, all fish in the control and treatment of 0.121, 0.240 mg/L were alive and appeared normal. Effects (Fish lying on side or back) occurred at concentrations of 0.503 mg/ and above. All fish were dead after 96h-exposure at concentration of 0.858, 1.27 mg/L. The results showed that under valid static test conditions, the 96 h-LC50 of the test substance to fish (Rare minnow, Gobiocypris rarus) was 0.500 mg/L, with 95% confidence limit of 0.420 mg/L ~ 0.600 mg/L (based on the measured concentration). The maximum tested concentration causing no mortality (96 h-LC0) was 0.240 mg/L. The minimum concentration causing 100% mortality (96 h-LC100) is 0.858 mg/L, i.e.:

96 h-LC50= 0.500 mg/L, 95% CI 0.420 ~ 0.600 mg/L (based on measured concentration);

96 h-LC0 = 0.240 mg/L (measured concentration);

96 h-LC100 = 0.858 mg/L (measured concentration).

Description of key information

Key value determined in GLP accredited laboratory, study according to the OECD guidelines for Testing of Chemicals, guideline No. 203: "Fish Acute Toxicity Test", Adopted 17 July, 1992, Council Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.1. "Acute Toxicity for Fish" and Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, 2000.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.91 mg/L

Additional information

A final test was performed based on results of a combined limit/range-finding test. Seven fish per group were exposed to an untreated control and nominal concentrations of 0.22, 0.46, 1.0, 2.2 and 4.6 mg/L. The total exposure period was 96 hours. Test solutions were renewed daily and samples for analytical determination of exposure concentrations were taken at the start and the end of the first and the last renewal.

 

Samples taken from all concentrations were analysed. The actual concentrations measured in the freshly prepared solutions were in agreement with nominal (89-100%).

Concentrations measured in the spent solutions at the end of the first refreshment period were between 44 and 90% of initial. The stability of the concentrations was increasing with the nominal dose. The actual concentrations in the spent solutions measured at the end of the last renewal period were at the level of 72-84% of initial.

 

Based on these results, the average exposure concentrations were 0.16, 0.33, 0.85, 1.8 and 4.0 mg/L in nominally of 0.22, 0.46, 1.0, 2.2 and 4.6 mg/L, respectively.

 

The study met the acceptability criteria prescribed by the study plan and was considered valid.

 

Under the conditions of the present study, the 96h-LC50was 0.91 mg/L based on average exposure concentrations (95% confidence interval between 0.69 and 1.2 mg/L).

Chinese testing laboratory.

Under static conditions, the acute toxicity of test substance (MLA-3202) to Rare minnow (Gobiocypris rarus) was conducted.

Nominal concentrations of 1 %, 2%, 4%, 6%, 8% and 10% stock solution were used in the definitive test.

In the range-finding test, 5 fish per treatment with no replicates were used. And 10 fish per treatment with no replicates were used in the definitive test. The fish were exposed for 96 hours to the test solution.

During the test period, the pH values of the control mediums and test mediums were between 7.72 and 7.97, the Dissolved Oxygen (DO) values varied from 75% ~ 97% of the air saturation at the test temperature, the temperature of the test mediums were maintained in the range of 22.8°C to 23.1°C, and the total hardness was in the range of 164 mg (CaC03)/L to 175 mg (CaC03)/L. All fishes in the control group were normal. With the same conditions, K1Cr201 was used as the positive control substance, and the resulting 24h-LC50 was 309 mg/L. So the study met the acceptability criteria prescribed by the protocol (The mortality of control :S 10%; pH: 6.0 ~ 8.5; dissolved oxygen concentration: >60% of the air saturation value; total hardness: 10 ~ 250 mg (CaC03)/L; temperature: (23±2)°C; 24h-LC50 of K1Cr201 in the range of 200 mg/L to 400 mg/L). Therefore, the test was considered valid.

During the test period, all fish in the control and treatment of 0.121, 0.240 mg/L were alive and appeared normal. Effects (Fish lying on side or back) occurred at concentrations of 0.503 mg/ and above. All fish were dead after 96h-exposure at concentration of 0.858, 1.27 mg/L. The results showed that under valid static test conditions, the 96 h-LC50 of the test substance to fish (Rare minnow, Gobiocypris rarus) was 0.500 mg/L, with 95% confidence limit of 0.420 mg/L ~ 0.600 mg/L (based on the measured concentration). The maximum tested concentration causing no mortality (96 h-LC0) was 0.240 mg/L. The minimum concentration causing 100% mortality (96 h-LC100) is 0.858 mg/L, i.e.:

96 h-LC50= 0.500 mg/L, 95% CI 0.420 ~ 0.600 mg/L (based on measured concentration);

96 h-LC0 = 0.240 mg/L (measured concentration);

96 h-LC100 = 0.858 mg/L (measured concentration).