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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
- Purity: 50.58 % of test substance in water.
Analytical monitoring:
yes
Details on test solutions:
Dilution water:
Source: Synthetic medium following NF EN ISO Norm 6341 from prepared from purified tap water (passed through activated charcoal and exchange resin and filtered at 0.22 µm)
Ca / Mg ratio: 4
Na / K ratio: 10
Oxygen content: « saturation »
Holding water different from dilution water: Yes, Elendt M7 supplemented with algae (specified as “Raphidocelis subcapitata”)
Test organisms (species):
Daphnia magna
Details on test organisms:
Strain: 5 (A) CAL laboratory culture
Age: <24 h
Breeding method: Clone, adults separated from juveniles by sieving
Kind of food: Algae (specified as “Raphidocelis subcapitata”)
Feeding of animals during test: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
ca. 20°C
Dissolved oxygen:
saturation
Salinity:
Ca / Mg ratio: 4
Na / K ratio: 10
Nominal and measured concentrations:
Nominal: 0, 0,0095, 0,015, 0,024, 0,039, 0,063 and 0.01 mg/L
Details on test conditions:
- Daphnids were exposed in a static test to 0, 0,0095, 0,015, 0,024, 0,039, 0,063 and 0.01 mg/L test substance, using 2 times 10 dahnids per concentration. - Analytical control at the end of the exposure demontrated that concentration levels remained constant during the exposure time of the study. However, concentrations measured differed from 62% at 0.015 mg/L, increasing with the increase of concentration to 97 and 96% for 0.063 and 0.1mg/L. Concentrations for the lower levels could not be analysed, as they were below the analytical detection limit of 7.3 µg/L. All concentrations given relate to the test substance as tested.
- The concentration-response relationship appears to be typical for this type of substance. The study can be considered as valid despite a few missing details (e.g. detail of volume of solution used, some information on the dilution water).
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.016 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.005 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility

48h-EC50 = 0.032 mg/L - 95% confidence levels 0.019 - 0.051 mg/L as nominal concentration.

Based on active substance:
48h-NOEC = 0.0048 mg a.i./L
48h-LOEC = 0.0075 mg a.i./L
48h-EC50 = 0.016 mg a.i./L
48h-EC100= 0.05 mg a.i./L

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.0048 mg a.i./L

Executive summary:

A study was conducted to determine the short-term toxicity to aquatic invertebrates of the test substance according to EU Method C.2, in compliance with GLP. Daphnia magna were exposed in a static test to nominal concentrations of 0, 0.0095, 0.015, 0.024, 0.039, 0.063 and 0.1 mg/L test substance, using 20 daphnids per concentration. Mobility was monitored throughout the experiment. Analytical dose verification was performed. Analytical control at the end of the exposure demonstrated that concentrations remained constant throughout the exposure period. However, measured concentrations differed by 62% at 0.015 mg/L, increasing to 97 and 96% at 0.063 and 0.1 mg/L. Concentrations for the lower levels could not be analysed as they were below the detection limit of 7.3 µg/L. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.0048 mg a.i./L (Thiebaud, 1999).

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 22, 2007 to February 24, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
but no impact on the study as the validity criteria for the study were met
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
but no impact on the study as the validity criteria for the study were met
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
yes
Remarks:
but no impact on the study as the validity criteria for the study were met
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report):ADBAC (i.e., benzyl C12-16 alkyl dimethyl ammonium chloride)
- Physical state: Clear liquid
- Analytical purity: 51%
- Lot/batch No.: DEGE108913
- Expiration date of the lot/batch: April 2007
- Stability under test conditions: Hydrolytically and photolytically stable under the conditions of this study and under standard laboratory conditions.
- Storage condition of test material: Ambient temperature
- Other: The test substance is known to be readily soluble, hydrolytically stable and has a very low vapour pressure.
Analytical monitoring:
yes
Details on sampling:
Two samples (5 mL) were taken from the freshly-prepared control and test media. After 48h, the contents of the test vessels from each group were pooled and further samples (5 mL) were taken for analysis. The samples were taken using a pipette and placed in glass vials; both of these items were pre-treated with a silanising agent (DMDCS) before use.

On each occasion, one of the samples was analysed and the other was stored in case further analysis was required. The reserve samples were stored frozen; it was not considered necessary to analyse these samples since the results obtained for the original samples were considered acceptable. Thus, the reserve samples were discarded.
Details on test solutions:
Preparation of test solutions:
The test organisms were maintained and the tests were conducted in softened Elendt M4 medium. The medium was prepared in deionised water produced by reverse osmosis. A stock solution for preparation of the test substance concentrations was prepared by diluting an aliquot of the test substance (19.6 mg) in the dilution medium (0.5 L) in a volumetric flask (1L). The contents of this flask were shaken vigorously before it was adjusted to volume with dilution medium. Aliquots (0.7, 1.2, 1.9, 3.1 or 5.5mL) of this stock solution were diluted with dilution medium to provide the test media.
- Mean water quality parameters, including temperature, pH, dissolved oxygen, total hardness and alkalinity remained within acceptable limits throughout the study.

Monitoring of the test substance concentration:
Duplicate samples of each freshly prepared control and test media were collected and the concentration of the test substance measured using an LC-MS method of analysis. The measured concentrations of the test substance ranged between 80 and 97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable, and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e. representative of exposure of the organisms to the nominal concentrations) and the EC50 value was determined using nominal concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
Original Daphnia broodstock was obtained from the Institute National de Recherche Chimique Appliqué (IRChA), France. Stock cultures of Daphnia magna were maintained in glass vessels containing approximately 0.8L of Elendt M4 culture medium in a temperature-controlled laboratory at nominally 20 ± 2ºC. A photoperiod of 16h light: 8h dark was maintained, with 60-min transition periods of subdued lighting at the beginning and end of each light phase. The light intensity of the test area was 611 lux.

- The culture medium was renewed three times each week.

-Cultures were fed daily with a suspension of the unicellular green algae, Pseudokirchneriella subcapitata, to provide nominally 0.1 to 0.2 mg carbon per daphnid, per day, except during the initial three days when a slightly lower ration was given (0.05 to 0.08 mg carbon per daphnid).
- Culture conditions ensure that the stock animals reproduce by pathenogenesis.
- The day before the start of the study, all juvenile Daphnia were removed from the laboratory cultures. The following morning, juveniles produced by gravid adult Daphnia were removed from the culture vessels and held in a separate holding vessel; these animals, which were less than 24h old, were used in the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 ± 1ºC
pH:
7.44-7.73
Dissolved oxygen:
99-104% air saturation value
Nominal and measured concentrations:
Nominal concentration: 0.007, 0.012, 0.019, 0.031, 0.052 mg a.i./L.
Details on test conditions:
Twenty juvenile daphnids (<24 h old) were exposed in each control and test group at the following nominal concentrations: 0.007, 0.012, 0.019, 0.031, and 0.052 mg a.i./L. The Daphnia were placed at random into glass vessels, four replicates of five animals per vessel, each containing approximately 150 mL of medium (resulting in a loading rate of 30 mL medium per organism). Each vessel was loosely covered with a watch glass. The temperature of the test area was 20 ± 1ºC. A 16-hour light: 8-hour dark photoperiod was maintained, with 60-minute periods of subdued lighting at the beginning and end of each phase. No supplementary aeration was employed and no feed was given during the exposure period.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.023 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 0.019-0.24 mg a.i./L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.016 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 0.0144-0.0177 mg a.i./L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.012 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
0.031 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
Test substance analysis:
The measured concentration of the test substance ranged between 80-97% of their nominal concentrations at the start of the test confirming that the intended exposure concentrations were achieved, so test results are expressed in terms of their nominal values. After 48h, the measured levels ranged between 30 and 42% of nominal. The test substance is known to be readily soluble, hydrolytically stable and has a very low vapour pressure. In addition, the test vessels were pre-treated with the test substance to prevent loss of the test substance to absorption/binding to the vessel walls. Therefore, the reduction of test substance concentration observed during the exposure period was attributed to absorption onto the test organism (i.e., representative of exposure of the organism to the nominal concentrations) and the EC50 value was determined using nominal concentrations.
Immobility: See the above results under 'Effect concentrations' and the table under 'any other information on results incl. tables'.


Results with reference substance (positive control):
The results from the most recent test performed prior to this study indicated that the reference substance 4h EC50 to Daphnia magna was 0.52 mg/L. This was within the range typically obtained in the laboratory (0.3 to 0.8 mg/L)
Reported statistics and error estimates:
Statistical analysis was performed using the SAFEStat LD50 application, SAS 8.2 (SAS Institute, 1999). Test results were expressed in terms of the nominal concentrations. The “no observed effect concentration” (NOEC) was derived by direct inspection of the data on the immobility of the animals. An incidence rate of more than 10% was considered to be significant.

Table 1. Immobilisation data

Concentration (mg a.i./l)

Percent Immobile at:

24 h

48 h

0.0

0

0

0.007

0

0

0.012

0

5

0.019

15

85

0.031

100

100

0.052

100

100

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 48h EC50 value of the test substance for the immobilisation of Daphnia magna was 0.016 mg a.i./L and the 48h NOEC was 0.012 mg a.i./L.
Executive summary:

A study was conducted to determine the short-term toxicity of the test substance to aquatic invertebrates according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010, in compliance with GLP. Toxicity was evaluated in Daphnia magna under static conditions with groups of twenty Daphnia. The test organisms were exposed for 48 h to nominal concentrations of 0, 0.007, 0.012, 0.019, 0.031 and 0.052 mg a.i./L test substance. The test media were prepared in softened Elendt M4 medium. Analytical dose verification was performed. Observations (for mobility) of the Daphnia were made after approximately 24 and 48 h. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.012 mg a.i./L (Jenkins, 2007).

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From May 09, 2003 to May 16, 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Remarks:
RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification. The aquatic invertebrates study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Qualifier:
according to
Guideline:
other: ISO/CD14669: "Determination of Acute Lethal Toxicity to Marine Copepods"
Deviations:
no
Qualifier:
according to
Guideline:
other: PARCOM ring test Protocol: "Acute Toxicity to the Marine Capepod Acartia tansa"
Deviations:
no
GLP compliance:
yes
Remarks:
GLP number: 41849
Specific details on test material used for the study:
- Composition of test material, percentage of components: ADBAC C12-14 content of 79.75%,
- Appearance: A clear light yellow liquid
- Stability: Stable
- Solubility: Soluble in water
- Specific gravity: 0.976 g/mL at 15°C
- Date received: May 05, 2003
- Storage: 20°C
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Preparation and application of test solution:
- Method: The test substance was soluble in water, so the sample preparation method for water soluble test substances was followed.
The test substance was dissolved in filtered and aerated seawater to prepare a stock solution with concentration corresponding to the highest test concentration. The stock solution was mixed in a 1L Erlenmeyer flask on a magnetic stirrer (~100 rpm) for 2-4h after covering the top to minimise the volatilization. Required concentrations were prepared form the stock solution.
- Controls: 4
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
Test organisms (species):
other: Acartia tonsa
Details on test organisms:
Test organism
- Common name: Calanoid copepod
- Source: Eggs of test organisms were obtained from University of Copenhagen, Laboratory of marine biology, Helsingør, Denmark
- Date of arrival of eggs: January 22, 2003
- Eggs were stored in refrigerator. Synchronious culture was used. The animals were cultivated in small batches.
- Age at study initiation: 14d
Test type:
not specified
Water media type:
saltwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20.0 - 21.1°C
Dissolved oxygen:
7.2 mg/L
Nominal and measured concentrations:
- Based on the results from the Skeletonema costatum range finding study, the following test concentrations (nominal whole product) were used for the definitive test: 0.05; 0.2; 0.8; and 3.2 mg/L.
- Reference substance (3,5-dichlorophenol) concentrations (nominal): 1.0 mg/L.
Details on test conditions:
Test system
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

Effect parameters measured: Mortality

Test concentrations:
- Range finding study: Dose levels selection was based on Skeletonema costatum range finding study
- Test concentrations: 0.05; 0.2; 0.8; and 3.2 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Calculated
Duration:
48 h
Dose descriptor:
LC10
Effect conc.:
0.37 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Calculated
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Calculated
Duration:
48 h
Dose descriptor:
other: LC90
Effect conc.:
0.43 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Calculated
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: Calculated
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: Calculated
Duration:
48 h
Dose descriptor:
LC10
Effect conc.:
0.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: Calculated
Details on results:
- Mortality of control: 0
- Mortality with test substance: Refer to Table 1
Results with reference substance (positive control):
- Reference substance study dates: May 14, 2003 to the May 16, 2003
- Results with reference substance valid? Yes
- Mortality: 55% after 48 h
Reported statistics and error estimates:
The LC50-value and 95% confidence intervals were calculated based from a Logistic regression model using JMP Program, version 3, 1998.

Test substance:

Results of the control and test substance groups are given below under tables:

Table 1. Test substance concentrations and mortality after 24 and 48h

Test concentration (mg/L)

Parallel

Number of organisms per vessel

Mortality (24 h)

Mortality (48 h)

Control

1

5

0

0

 

2

5

0

0

 

3

5

0

0

 

4

5

0

0

0.05

1

5

0

0

 

2

5

0

0

 

3

5

0

0

 

4

5

0

0

0.2

1

5

0

0

 

2

5

0

0

 

3

5

0

0

 

4

5

0

0

0.8

1

5

5

5

 

2

5

5

5

 

3

5

5

5

 

4

5

5

5

3.2

1

5

5

5

 

2

5

5

5

 

3

5

5

5

 

4

5

5

5

 

Table 2. Calculation of LC10, LC50and LC90-values after 24 and 48h (The 95% confidence range could not be calculated)

24 and 48h

Effect level

mean (log)

LC10

-0.43

LC50

-0.40

LC90

-0.37

Effect level

mean (inverse log)

LC10

0.37

LC50

0.40

LC90

0.43

 

Summary of the results:

The LC50-value for the test substance was found to be 0.40 mg/L after 24 and 48 h. The 95% confidence range could not be calculated.

The NOEC-, LC10- and LC90-values after 24 and 48h were 0.2, 0.37 and 0.43 mg/L, respectively.

Reference substance:

The lethality with 1.0 mg/L 3,5-dichlorophenol was 55% after 48 h.

Validity criteria fulfilled:
yes
Conclusions:
Under the study condictions, the 48 h NOEC, LC10, LC50 and LC90 were 0.2, 0.37, 0.40 and 0.43 mg/L (equivalent to 0.2, 0.3, 0.3 and 0.3 mg a.i./L), respectively
Executive summary:

A study was conducted to determine the toxicity to aquatic invertebrates of the read across substance, benzyl-C12-14-alkyldimethylammonium chlorides (C12-14 ADBAC), according to ISO/CD14669 and PARCOM ring test Protocol, in compliance with GLP. The experiment assessed the effects on mortality of a 48 h exposure of Acartia tonsa. The test was performed on 14 d old organisms. Nominal test substance concentrations were 0, 0.05, 0.2, 0.8 and 3.2 mg/L, equivalent to 0, 0.04, 0.2, 0.6 and 2.6 mg a.i./L based on a purity of 79.75%. The reference substance 3,5-dichlorophenol was included in the study. Under the study conditions, the 48 h NOEC, LC10, LC50 and LC90 were 0.2, 0.37, 0.40 and 0.43 mg/L (equivalent to 0.2, 0.3, 0.3 and 0.3 mg a.i./L), respectively (Haug, 2003).

Description of key information

Based on mobility and mortality, the 48 h EC50 value for Daphnia magna (freshwater) and Acartia tonsa (marine water) due to the test substance were determined to be 0.016 mg/L and 0.32 mg a.i./L (nominal) respectively.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.016 mg/L
EC50/LC50 for marine water invertebrates:
0.32 mg/L

Additional information

Study 1. A study was conducted to determine the short-term toxicity to aquatic invertebrates of the test substance according to EU Method C.2, in compliance with GLP. Daphnia magna were exposed in a static test to nominal concentrations of 0, 0.0095, 0.015, 0.024, 0.039, 0.063 and 0.1 mg/L test substance, using 20 daphnids per concentration. Mobility was monitored throughout the experiment. Analytical dose verification was performed. Analytical control at the end of the exposure demonstrated that concentrations remained constant throughout the exposure period. However, measured concentrations differed by 62% at 0.015 mg/L, increasing to 97 and 96% at 0.063 and 0.1 mg/L. Concentrations for the lower levels could not be analysed as they were below the detection limit of 7.3 µg/L. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.0048 mg a.i./L (Thiebaud, 1999).

Study 2. A study was conducted to determine the short-term toxicity of the test substance to aquatic invertebrates according to OECD Guideline 202, EU Method C.2 and US EPA OPPTS 850.1010, in compliance with GLP. Toxicity was evaluated in Daphnia magna under static conditions with groups of twenty Daphnia. The test organisms were exposed for 48 h to nominal concentrations of 0, 0.007, 0.012, 0.019, 0.031 and 0.052 mg a.i./L test substance. The test media were prepared in softened Elendt M4 medium. Analytical dose verification was performed. Observations (for mobility) of the Daphnia were made after approximately 24 and 48 h. Under the conditions of the study, the 48 h EC50 was 0.016 mg a.i./L and the 48 h NOEC was 0.012 mg a.i./L (Jenkins, 2007).

Study 3. A study was conducted to determine the toxicity to aquatic invertebrates of the read across substance, benzyl-C12-14-alkyldimethylammonium chlorides (C12-14 ADBAC), according to ISO/CD14669 and PARCOM ring test Protocol, in compliance with GLP. The experiment assessed the effects on mortality of a 48 h exposure of Acartia tonsa. The test was performed on 14 d old organisms. Nominal test substance concentrations were 0, 0.05, 0.2, 0.8 and 3.2 mg/L, equivalent to 0, 0.04, 0.2, 0.6 and 2.6 mg a.i./L based on a purity of 79.75%. The reference substance 3,5-dichlorophenol was included in the study. Under the study conditions, the 48 h NOEC, LC10, LC50 and LC90 were 0.2, 0.37, 0.40 and 0.43 mg/L (equivalent to 0.2, 0.3, 0.3 and 0.3 mg a.i./L), respectively (Haug, 2003).