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Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 235 (Chironomus sp., Acute Immobilisation Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At exposure initiation and termination, one sample from each test concentration and control was collected.
Vehicle:
yes
Remarks:
Solvent dimethylformamide was used
Details on test solutions:
A 500 μg/mL primary stock solution was prepared by placing an appropriate amount of substance in a glass volumetric flask and bringing it to full volume with dimethylformamide (DMF). Secondary stock solutions at lower concentrations were then prepared by further diluting the primary stock solution. These stock solutions were all clear and colourless with no visible undissolved test substance. Stock solutions were then diluted with appropriate volumes of dilution water (laboratory well water) to obtain the test solutions at the correct nominal test concentrations.
Test organisms (species):
Chironomus riparius
Details on test organisms:
Dipteran midge larvae (Chironomus riparius) were selected as test organism, because they are widely distributed globally, important in aquatic food webs, easily cultured and have a relatively short life cycle. The larvae used in the test came from own cultures of the performing laboratory.
Midge were maintained in laboratory well water that was supplemented with dechlorinated town well water.
Prior to exposure initiation, egg masses were isolated individually from the main culture tanks from the same brood stock, removed from culture aquaria and each individual egg mass was placed in a 30 mL plastic cup with about 25 mL of culture water. The isolated egg masses were observed daily until hatching was complete. The larvae were derived from a healthy stock. During the period of larval holding, the reserved population was examined for mortality and general behaviour. Dissolved oxygen was in the range from 9.1 to 9.8 mg/L and temperature was in the range from 20 to 21 °C prior to exposure initiation.
Culture midge were fed finely ground flaked fish food suspension at a concentration of 100 mg/mL daily. During the holding and acclimation period, larvae were fed daily a finely ground flaked fish food suspension at a concentration of 10 mg/mL. Midge were not fed during the 48-hour exposure period. Fish food was analysed periodically to confirm the absence of pesticides, PCBs and toxic metals.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
76 mg/L as CaCO3
Test temperature:
19 to 21 °C
pH:
7.1 to 7.6
Dissolved oxygen:
8.9 to 10 mg/L (99 to 110% of saturation)
Salinity:
Total alkalinity of 20 mg/L as CaCO3
Conductivity:
670 μS/cm
Nominal and measured concentrations:
Nominal: 0, 1.0, 2.4, 6.4, 16, 40, 100 ng/L
Mean measured average: <0.5, 1.1, 2.3, 5.6, 14, 38, 96 ng/L
Details on test conditions:
Tests were conducted in 250 mL glass beakers, which were chemically cleaned before use. The test volume was maintained at 200 mL. Four replicates were used per test concentration, negative control, solvent control and positive control. Five midges were added to each test vessel.
Test vessels were maintained at a temperature of 20 ± 2 °C with a 16 hour light to 8 hour darkness photoperiod at a light intensity of 660 to 930 lux.
Reference substance (positive control):
yes
Remarks:
Potassium chloride was used as positive control substance.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
15 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
No adverse effects or immobilisation were observed in the negative control. Mortality was 5% among organisms exposed to the solvent control, which is within the acceptable range of 15% for control populations.
Results with reference substance (positive control):
The 48-hour EC50 value was 3.6 g/L with the positive reference substance, which was within the expected range for the exposure of test organisms to potassium chloride.
Reported statistics and error estimates:
95% confidence interval of EC50 value: 10 to 22 ng/L
Validity criteria fulfilled:
yes
Conclusions:
The 48-hour EC50 value in the acute toxicity study with midge was 15 ng/L, based on immobilisation of test organisms.
Executive summary:

The acute toxicity of the substance to a freshwater midge (Chironomus riparius) was studied under GLP to OECD TG 235 over a period of 48 hours. Stock solutions were prepared by dissolving appropriate amounts of test substance in the solvent dimethylformamide. These stock solutions were then diluted with water to obtain the test solutions at appropriate nominal test concentrations of 1, 2.4, 6.4, 16, 40 and 100 ng/L. Quality parameters of the dilution water were: total hardness and total alkalinity as CaCO3 76 mg/L and 20 mg/L, pH value 7.0, conductivity 670 μS/cm, total organic carbon content 1.2 mg/L. Four replicates were tested per test concentration alongside with negative controls, solvent controls and positive controls (potassium chloride) in 250 mL glass beakers containing a volume of 200 mL test solution. Five midge were added to each test vessel. Test organisms were obtained from healthy breeding stocks maintained under appropriate conditions in the test laboratory. Test vessels were kept at a temperature of 20 ± 2 °C with a 16 hour light to 8 hour darkness photoperiod at a light intensity of 660 to 930 lux. Test concentrations were confirmed by a suitable analytical procedure showing satisfactory precision and recovery using liquid chromatography with tandem mass spectrometry detection. The mean measured concentrations over the exposure period of 48 hours were 1.1, 2.3, 5.6, 14, 38 and 96 ng/L, the pH value ranged from 7.1 to 7.6, the dissolved oxygen concentration ranged from 8.9 to 10 mg/L and the temperature was 19 to 21 °C during the exposure period. Following 48 hours of exposure, a concentration dependent increase in the immobilisation of test midge was observed. The 48-hour EC50 value was determined to be 15 ng/L, with a 95% confidence interval  of 10 to 22 ng/L. The validity criteria of the study were fulfilled, i.e. not more than 15% of the larvae in the control were immobilised or showed signs of disease or stress, the dissolved oxygen concentration was >3 mg/L in the control and test vessels, and the results of the positive control confirmed the suitability and susceptibility of the test system.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 Jun 2018 to 12 Jun 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1035 (Mysid Acute Toxicity Test)
Version / remarks:
2016
Deviations:
no
Remarks:
see Deviations from the Protocol in 'Any other information on materials and methods incl. tables'.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At exposure initiation (0 hour), the 48-hour interval (aged and new solutions), and exposure termination (96 hours), one sample from each treatment level and control was collected and analyzed for the test substance concentration. New solution samples were collected at the 0- and 48-hour intervals and aged solution samples were collected at the 48- and 96-hour intervals. Each sample was collected from the approximate midpoint of the test vessel using a pipet. At exposure initiation and the 48-hour interval, samples were removed from the intermediate mixing vessels of each treatment level and control solution prior to division into the replicate vessels. At the 48-hour interval and exposure termination, samples were removed from composited replicate solutions of each individual treatment level and the controls. Archive samples were also removed in the same manner on these exposure days and stored frozen as a contingency in case further analysis of samples was deemed needed. Samples of the stock solutions were also analyzed at the 0-hour interval.
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
Prior to exposure initiation, a 100 µg/mL primary stock solution was prepared by bringing 0.0199 g of the test substance to a final volume of 200 mL with dimethylformamide (DMF). The primary stock solution was mixed with inversions of the volumetric flask and was observed to be clear and colorless with no visible undissolved test substance following preparation. The preparation scheme is provided in Table 1 in "Any other information on materials and methods incl. tables".
All secondary stock solutions were observed to be clear with and colorless with no visible undissolved test substance following mixing by inversions of the flask. The preparation scheme of the exposure solution is provided in Table 2 in "Any other information on materials and methods incl. tables".
All exposure solutions were mixed using a glass rod for approximately one minute. All solutions were observed to be clear and colorless, with no visible undissolved test substance following preparation. These solutions were prepared twice as described above. The first set of solutions was used to condition the intermediate preparation beakers and exposure vessels prior to exposure initiation. These solutions were discarded. Freshly-prepared exposure solutions for each treatment level and control were prepared at the 48-hour renewal interval. The control vessels were maintained under the same conditions as the treatment level solutions but contained no test substance. Each test vessel was labeled with the study number, replicate designation, and concentration.
Test organisms (species):
Americamysis bahia (previous name: Mysidopsis bahia)
Details on test organisms:
TEST ORGANISM
- Common name: Mysids
- Source: Obtained from laboratory cultures continuously maintained at the test facility (Mysids used for this test were obtained by isolating sexually mature adults from the culture vessels 24 hours prior to exposure initiation. )
- Age at study initiation: ≤24 hours old

ACCLIMATION
- Acclimation period: 2 Weeks
- Acclimation conditions: Cultured in one of several 76 L glass aquaria with a closed-loop recirculating filtration system providing natural, filtered seawater to the aquaria.
- Salinity: 21 to 23‰
- Temperature: 21 to 23‰
- Photoperiod: 16 hours of light and 8 hours of darkness
- Mysids were maintained under conditions similar to those in the definitive test.
- Representative samples of the food source were analyzed periodically for the presence of pesticides, PCBs, and toxic metals. None of these compounds have been detected at concentrations that are considered toxic in any of the food samples analyzed, in agreement with ASTM (2007) standard practice. Based on these analyses, the food sources were considered to be of acceptable quality since all analyte concentrations were below limits of concern.

FEEDING
- Type of food: Live brine shrimp (Artemia salina) nauplii
- Feeding frequency: Twice daily prior to the exposure and once daily during the exposure
Test type:
semi-static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 - 27 °C
pH:
7.6 - 7.8
Dissolved oxygen:
4.7 - 7.3 mg O2/L
Salinity:
19 - 20‰
Nominal and measured concentrations:
- Nominal concentration: 0 (negative control), 0 (solvent control), 0.0031, 0.0063, 0.013, 0.025, 0.050, and 0.10 µg/L
- Measured concentration: < LOQ (negative control), < LOQ (solvent control), 0.0030, 0.0064, 0.013, 0.026, 0.053, and 0.12 µg/L, respectively. See Table 3 in 'Any other information on materials and methods incl tabels'.
Details on test conditions:
TEST SYSTEM
- Test vessel: 600 mL glass beakers
- Volume of solution: Approximately 500 mL of exposure solution
- No. of organisms per vessel: 10
- No. of vessels per concentration: 2
- No. of vessels per control: 2
- No. of vessels per vehicle control: 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water used during this study was from the same source as the water used during culture and holding. Aged, dilute, filtered natural seawater was prepared by collecting natural seawater from the Cape Cod Canal, Bourne, Massachusetts. The water was collected at about 1 to 4 meters offshore and a depth of approximately 0.5 meters. The seawater was then transferred by a pump (fiberglass reinforced thermoplastic housing) through polyvinyl chloride (PVC) pipes and transported to the laboratory in a 6080 L polyethylene holding tank. In the laboratory, the seawater was diluted to a salinity of 20 ± 3‰ with laboratory well water, filtered through 20 µm, 5 µm, and 1 µm polypropylene core filters.
- Total organic carbon (TOC): Representative samples of the dilution water were analyzed monthly for TOC concentration. The TOC concentration in the dilution water was 1.6 mg/L for June 2018. The TOC concentration falls within the recommendation of the guideline (i.e., < 2.0 mg/L).
- Representative samples of the dilution water were analyzed periodically for the presence of pesticides, PCBs, and toxic metals.None of these compounds have been detected at concentrations that are considered toxic in any of the food samples analyzed, in agreement with ASTM (2007) standard practice.
- Intervals of water quality measurement: The pH, dissolved oxygen concentration, temperature, and salinity were measured daily in each test vessel. Temperature was continuously monitored throughout the study in a satellite vessel located in the environmental chamber.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours of darkness. Electronic timers were used to provide a 15- to 30-minute transition period.
- Light intensity and location: 640 to 980 lux (59 to 91 footcandles) at the surface of the solution

EFFECT PARAMETERS MEASURED
Biological observations including sublethal effects, number of mortalities and observations of the physical characteristics of the exposure solutions (e.g., precipitated materials, cloudiness, etc.) were recorded at exposure initiation and at each subsequent 24-hour interval until exposure termination (96 hours), if applicable. The criteria used for determining death were the lack of movement, absence of respiratory movement, and lack of reaction to gentle prodding. Dead organisms were removed and discarded at each observation interval.

RANGE-FINDING STUDY
- Study design: During the preliminary exposure, mysids were exposed under static-renewal conditions. All exposure solutions were observed to be clear and colorless with no visible undissolved test substance following preparation. Two test vessels containing 10 mysids each were established for each treatment level and the controls. The test vessels were 1.0 L glass beakers each containing approximately 900 mL of exposure solution.
- Nominal concentrations: 0.00010, 0.0010, 0.010, 0.10, and 1.0 µg/L, a negative control, and a solvent (DMF) control
- Results used to determine the conditions for the definitive study: Following 96 hours of exposure, mortality of 5, 10, 20, 100, and 100% was observed among mysids exposed to the 0.00010, 0.0010, 0.010, 0.10, and 1.0 µg/L treatment levels, respectively. No mortalities or adverse effects were observed the negative control or solvent (DMF) control. Based on these results and consultation with the Study Sponsor, nominal concentrations of 0.0031, 0.0063, 0.013, 0.025, 0.050, and 0.10 µg/L were selected for the definitive exposure.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.018 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other:
Remarks:
95% C.L.: 0.013 - 0.026 µg/L
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.001 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
An overview of the reusults is provided in Table 5 and Table 6 'Any other information on results incl tables'.
Following 96 hours of exposure, 100% mortality was observed among mysids exposed to the 0.026, 0.053, and 0.12 µg/L mean measured treatment levels. Mortality of 5% was observed among mysids exposed to the solvent control and the 0.0030 µg/L mean measured treatment level. No mortalities or sublethal effects were observed among mysids exposed to the remaining treatment levels tested (0.0064 and 0.013 µg/L mean measured concentrations) or the negative control. ASTM (2007) recognizes the limitations of acute toxicity testing, i.e., response less than or equal to 10% is allowable in a control population and is considered within the expected range of naturally occurring variability. Therefore, the mortality (5%) observed in the solvent control and the 0.0030 µg/L mean measured concentration treatment level is considered to be biological variability and not an adverse response from exposure to the test substance.

Table 5. Mean Measured Concentrations Tested, Corresponding Cumulative Percent and Number of Mortalities, and Observations Made during the 96-Hour Static-Renewal Exposure of Mysids (Americamysis bahia) to the test substance.

Mean Measured Concentration

(µg/L)

Cumulative Percent Mortalitya

24-Hour

48-Hour

72-Hour

96-Hour

A

B

Mean

A

B

Mean

A

B

Mean

A

B

Mean

 

Negative Control

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

(0)

(0)

 

(0)

(0)

 

(0)

(0)

 

(0)

(0)

 

--

--

 

--

--

 

--

--

 

--

--

 

Solvent Control

0

10

5

0

10

5

0

10

5

0

10

5b

(0)

(1)

 

(0)

(1)

 

(0)

(1)

 

(0)

(1)

 

--

--

 

--

--

 

--

--

 

(0)

--

 

0.0030

0

0

0

0

10

5

0

10

5

0

10

5b

(0)

(0)

 

(0)

(1)

 

(0)

(1)

 

(0)

(1)

 

--

--

 

--

--

 

--

--

 

--

--

 

0.0064

0

0

0

0

0

0

0

0

0

0

0

0

(0)

(0)

 

(0)

(0)

 

(0)

(0)

 

(0)

(0)

 

--

--

 

--

--

 

--

--

 

--

--

 

0.013

0

0

0

0

0

0

0

0

0

0

0

0

(0)

(0)

 

(0)

(0)

 

(0)

(0)

 

(0)

(0)

 

--

--

 

--

--

 

--

--

 

--

--

 

0.026

0

0

0

50

30

40

90

90

90

100

100

100

(0)

(0)

 

(5)

(3)

 

(9)

(9)

 

(10)

(10)

 

--

--

 

L

L

 

L

L

 

NA

NA

 

0.053

10

0

5

90

90

90

100

100

100

100

100

100

(1)

(0)

 

(9)

(9)

 

(10)

(10)

 

(10)

(10)

 

--

4L

 

L

B

 

NA

NA

 

NA

NA

 

0.12

0

10

5

100

100

100

100

100

100

100

100

100

(0)

(1)

 

(10)

(10)

 

(10)

(10)

 

(10)

(10)

 

L

L

 

NA

NA

 

NA

NA

 

NA

NA

 

-- = No sublethal effects observed; L = lethargic; B = at bottom of exposure vessel; NA = Not Applicable

a The actual number of mortalities is presented in parentheses.

b ASTM (2007) recognizes the limitations of acute toxicity testing, i.e., response less than or equal to 10% is allowable in a control population and is considered within the expected range of naturally occurring variability. Therefore, the mortality (5%) observed in the solvent control and the 0.0030 µg/L mean measured concentration treatment level is considered to be biological variability and not an adverse response from exposure to the test substance.

Table 6. The LC50 Values, Corresponding 95% Confidence Intervals, and No-Observed-Effect Concentration (NOEC) Established during the 96-Hour Static-Renewal Exposure of Mysids (Americamysis bahia) to the test substance

Based on Mean Measured Concentrations

 

Time Interval

LC50

(µg/L)

95% Confidence Interval

(µg/L)

 

Lower

Upper

24-Houra

>0.12

NAb

NA

48-Hourc

0.031

0.025

0.037

72-Hourc

0.020

0.018

0.022

96-Hourd

0.018

0.013

0.026

96 Hour NOEC = 0.013 µg/L

a LC50 value empirically estimated to be greater than the highest mean measured concentration tested; therefore, 95% confidence interval could not be determined.

b NA = Not Applicable

c LC50 value and the corresponding 95% confidence interval was determined by Trimmed Spearman-Kärber Estimates.

d LC50 value and the corresponding 95% confidence interval was determined by Binomial Graphical Estimates

Validity criteria fulfilled:
yes
Conclusions:
Based on the mean measured concentrations, the 96-hour LC50 value was 0.018 µg/L, with a 95% confidence interval of 0.013 to 0.026 µg/L.
Executive summary:

To determine the 96-hour acute toxicity of the test substance to the mysid (Americamysis bahia), the exposure was conducted under static-renewal conditions following OCSPP Guideline 850.1035. The test was in compliance with GLP. Two replicate vessels, each containing ten mysids, were established for each treatment level, negative control, and solvent control. Nominal concentrations for the treatment levels tested were 0.0031, 0.0063, 0.013, 0.025, 0.050, and 0.10 µg/L (0.0030, 0.0064, 0.013, 0.026, 0.053, and 0.12 µg/L mean measured concentrations). Exposure vessels were maintained in a temperature-controlled environmental chamber at 25 ± 1 °C with a 16 hour light: 8 hour dark photoperiod at a light intensity range of 640 to 980 lux (59 to 91) footcandles. Test vessels were examined at 0, 24, 48, 72, and 96 hours of exposure as follows: dead mysids were recorded and removed, biological observations, including adverse effects (i.e., observations of stress, abnormal behavioral activity, and mortality) of the exposed mysids and observations of the physical characteristics of the exposure solutions (i.e., presence of precipitate, cloudiness, etc.) were made and recorded. Exposure and control solutions were renewed at the 48-hour interval to maintain test concentrations. Effects for this study were based on death, defined as the lack of movement, absence of respiratory movement, and lack of reaction to gentle prodding.

Following 96 hours of exposure, 100% mortality was observed among mysids exposed to the 0.026, 0.053, and 0.12 µg/L mean measured treatment levels. Mortality of 5% was observed among mysids exposed to the solvent control and the 0.0030 µg/L mean measured treatment level. No mortality or sublethal observations were observed among mysids exposed to the remaining treatment levels tested (0.0064 and 0.013 µg/L mean measured concentrations) or the negative control. ASTM (2007) recognizes the limitations of acute toxicity testing, i.e., response less than or equal to 10% is allowable in a control population and is considered within the expected range of naturally occurring variability. Therefore, the mortality (5%) observed in the solvent control and the 0.0030 µg/L mean measured concentration treatment level is considered to be biological variability and not an adverse response from exposure to the test substance.

Based on the mean measured concentrations, the 96-hour LC50 value was determined using Binomial Graphical Estimates to be 0.018 µg/L, with a 95% confidence interval of 0.013 to 0.026 µg/L. The NOEC was empirically estimated to be 0.013 µg/L.

Description of key information

Freshwater, 48-h EC50 = 0.015 µg/L (corresponding 95% confidence intervals of 0.01 to 0.022 µg/L), Chironomus riparius, Immobilization, OECD TG 235, Shaw 2019


Marine water, 96-h LC50 = 0.018 µg/L (corresponding 95% confidence intervals of 0.013 to 0.026 µg/L), Americamysis bahia, mortality, OCSPP TG 850.1035, Shaw 2019

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
EC50
Effect concentration:
0.015 µg/L

Marine water invertebrates

Marine water invertebrates
Dose descriptor:
LC50
Effect concentration:
0.018 µg/L

Additional information

Freshwater


The acute toxicity of the substance to freshwater invertebrates was tested under GLP and to the relevant OECT test guidelines 202 and 235. Three reliable and valid studies conducted with water fleas (Daphnia magna), midge (Chironomus riparius) and amphipods (Hyalella azteca) demonstrated that the substance exhibits significant short-term toxicity to freshwater invertebrates. The freshwater midge Chironomus riparius was most sensitive to exposure to the substance and the corresponding acute aquatic toxicity study resulted in a 48-hour EC50 value of 0.015 µg/L (corresponding 95% confidence intervals of 0.01 to 0.022 µg/L), based on the immobilisation of midge at the end of the exposure period. Freshwater amphipods were slightly less susceptible to the substances, as indicated by the 96-hour LC50 value of 0.041 µg/L. The water flea (Daphnia magna), which is the preferred organism to test the acute toxicity to freshwater invertebrates und REACH, was least sensitive to the substance, and the respective 48-hour EC50 value was 0.52 mg/L.


Marine water


The acute toxicity of the substance to marine invertebrates was tested under GLP and to US EPA OCSPP Guideline 850.1035. A reliable and valid study conducted with mysids (Americamysis bahia) demonstrated that the substance exhibits significant short-term toxicity to marine invertebrates. The 96-hour LC50 value of 0.018 µg/L (corresponding 95% confidence intervals of 0.013 to 0.026 µg/L), based on the mortality of mysids at the end of the exposure period. The tested marine invertebrate species showed a sensitivity to the test substance that was comparable to that of the most susceptible freshwater species.