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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:
31 March 2016 - 19 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Treated at nominal WAF loading rates.
Test organisms (species):
Daphnia magna
Details on test organisms:
The cladoceran, Daphnia magna, was selected as the test species for this study. This species is representative of an important group of aquatic invertebrates and was selected for use in the test based upon past history of use in the laboratory. Daphnids used in the test were <24-hour old neonates obtained from cultures maintained by Wildlife International of Easton, Maryland. Identification of the species was verified by the supplier of the original stock culture. Adult daphnids were cultured in water from the same source and at approximately the same temperature as used during the test. During the 2-week period immediately preceding the test, water temperatures in the cultures ranged from 19.4 to 20.6ºC, the pH of the water ranged from 8.1 to 8.4, and the dissolved oxygen concentrations were ¿7.7 mg/L (¿86% of saturation). The four adult daphnids used to supply neonates for the test were held for 27 days prior to collection of the juveniles for testing, and had each produced at least one previous brood. Adult daphnids in the culture had produced an average of at least three young per adult per day over the 7-day period prior to the test. The adults showed no signs of disease or stress, no ephippia were produced during the holding period, and mortality in the culture stock was <20% in the two-day period prior to test initiation.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Observations were made approximately 5, 24 and 48 hours after test initiation.
Hardness:
The hardness was 152 mg/L as CaCO3.
Test temperature:
The temperature was in the range of 20.1 - 20.9°C.
pH:
The pH was in the range of 8.1 - 8.5.
Dissolved oxygen:
The dissolved oxygen content was in the range of 8.1-8.5 mg/L
Salinity:
not applicable
Conductivity:
348 µS/cm
Nominal and measured concentrations:
Nominal WAF loading rates selected for use in this study were 0.41, 1.2, 3.7, 11, 33 and 100 mg/L.
Details on test conditions:
The test systems were illuminated using fluorescent tubes that emit wavelengths similar to natural sunlight. The lights were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness. A 30-minute transition period of low light intensity was
provided when lights went on and off to avoid sudden changes in light intensity. Light intensity was measured at the water surface of one representative test chamber at the beginning of the test using a SPER Scientific Model 840006 light meter.

The test was conducted at a target water temperature of 20 ± 1¿C. Temperature was measured in each test chamber at the beginning of the test, at approximately 24 hours during the test, and at the end of the test using a digital thermometer. Water temperature also was monitored continuously in a container of water placed adjacent to the test chambers using a validated environmental monitoring system (AmegaView Central Monitoring System). The system measurements were calibrated prior to exposure initiation with a digital thermometer.

Dissolved oxygen and pH were measured in each test chamber at the beginning of the test, at approximately 24 hours during the test, and at the end of the test. Dissolved oxygen was measured using a Thermo Orion Star A213 dissolved oxygen meter, and measurements of pH were made using a Thermo Orion Dual Star pH/ISE meter.

Hardness, alkalinity, specific conductance and total organic carbon (TOC) in the dilution water at the beginning of the test were measured. Hardness and alkalinity measurements were made by titration based on methods in Standard Methods for the Examination of Water and Wastewater.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
All daphnids in the negative control group appeared normal throughout the test. All daphnids in the 0.41 mg/L treatment group also appeared normal throughout the test, with no immobile daphnids or overt signs of toxicity observed. Percent immobility in the 1.2, 3.7, 11, 33 and 100 mg/L treatment groups at test termination was 0, 0, 5, 0 and 5%, respectively. Signs of toxicity observed among the surviving daphnids in the 1.2, 3.7, 11, 33 and 100 mg/L treatment groups at test termination included lethargy.

See attached background material

Validity criteria fulfilled:
yes
Conclusions:
Based on the nominal WAF loading rates, the 48-hour EL50 value (the theoretical WAF loading rate that produced 50% immobility) was determined to be > 100 mg/L, the highest loading rate tested. The NOEL was determined to be 0.41 mg/L.
Executive summary:

The study was conducted in accordance with OECD Guideline 202 in order to determine toxicity of the test substance to the Daphnid (Daphnia Magna) in a 48 hour static test.

 

WAFs were prepared in water at 0.41, 1.2, 3.7, 11, 33, and 100 mg/L.

All daphnids in the negative control group appeared normal throughout the test. All daphnids in the 0.41 mg/L treatment group also appeared normal throughout the test, with no immobile daphnids or overt signs of toxicity observed. Percent immobility in the 1.2, 3.7, 11, 33 and 100 mg/L treatment groups at test termination was 0, 0, 5, 0 and 5%, respectively. Signs of toxicity observed among the surviving daphnids in the 1.2, 3.7, 11, 33 and 100 mg/L treatment groups at test termination included lethargy.

 

Based on the nominal WAF loading rates, the 48-hour EL50value (the theoretical WAF loading rate that produced 50% immobility) was determined to be > 100 mg/L, the highest loading rate tested. The NOEL was determined to be 0.41 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2002 - 3 July 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Amber solid
Received: 13-Sep-99
Expiration date: 30-Sep-04
Storage Room Temperature
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the water-accommodated fraction (WAF) and control solution on Day 0 and on a composite of the replicates at termination (Day 2). The samples were analyzed for total organic carbon (TOC).
Vehicle:
no
Details on test solutions:
Individual treatments were prepared by adding the appropriate amount of test substance to 4.0 L of laboratory dilution water in glass aspirator bottles. The test substance is a solid, it was ground using a mortar and pestle prior to WAF preparation; none of the particles appeared to be larger than 1 mm from a visual determination. The WAFs were prepared by weighing the substance onto plastic pans and then adding the substance to the dilution water in the mixing vessels. The mixing vessels were sealed with Teflon® covered neoprene stoppers. The mixtures were stirred at <10% vortex for 41 hours and 30 minutes on magnetic stirplates with Teflon® coated stirbars at room temperature. During mixing, the treatments appeared clear / colorless with approximately 95% of the test substance at the surface and approximately 5% of the test substance floating in solution. After mixing, the mixtures were allowed to settle for 4 hours and 5 minutes in a water bath at 20.0¿C, before the aqueous portions were removed through the outlet at the bottom of the vessels. After settling the mixtures appeared clear / colorless with the test substance on the bottom of the mixing vessel. Four replicates were prepared by filling the test chambers with approximately 140 mL of the WAFs. Four replicates of the control were prepared in the same manner using reconstituted water.
Test organisms (species):
Daphnia magna
Details on test organisms:
Cultured at the test facility. Original culture supplied by Aquatic Biosystems, Inc., Fort Collins, CO. Starter culture received 11-Apr-02.

Eight daphnids are kept in 1-liter glass culture beakers with approximately 800 mL of reconstituted water (study dilution water), supplemented with vitamin B12 and selenium. The culture chamber is maintained at 20 ± 1°C under a 16 hour light 8 hour dark photoperiod (161 - 215 Lux). Day 0 cultures are started daily (at least five days per week) using ten <24 hour old neonates from culture beakers between 12 and 18 days old, exhibiting =20% adult mortality and <10% neonate mortality. Cultures are transferred to fresh reconstituted water on regular intervals to ensure that <24 hour old neonates are available for studies and to start new cultures.

Cultures of Daphnia magna Strauss are fed Pseudokirchneriella subcapitata (approximately 4.5 x 105 cells/mL) and 4.0 mL of a yeast / salmon starter / wheat grass (YTC) mixture per 800 mL daily (five days per week at a minimum). YTC and algae were supplied by Aquatic Biosystems, Inc., Fort Collins, CO.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Observations for immobilisation of the daphnids were made on each replicate at 24 and 48 hours (¿ 1 hour). Immobilisation is defined as the lack of swimming ability or movement within 15 seconds after gentle agitation of the test container. In addition, observations for normal or abnormal daphnid behavior or appearance were made.
Hardness:
The hardness as CaCo2 = 88 mg/L
Test temperature:
Mean test temperature: 19.7°C (S.D. = 0.4).
pH:
The pH = 7.7
Dissolved oxygen:
The Dissolced Oxygen = 9.0 mg/L
Salinity:
not applicable
Conductivity:
The specific Conductance = 305 µmhos
Nominal and measured concentrations:
Nominal = 101 mg/L
Mean TOC = 0.7033 mg/L

Details on test conditions:
Preparation and Administration of Test Substance - Individual treatments were prepared by adding the appropriate amount of test substance to 4.0 L of laboratory dilution water in glass aspirator bottles. The test substance is a solid, it was ground using a mortar and pestle prior to WAF preparation; none of the particles appeared to be larger than 1 mm from a visual determination. The WAFs were prepared by weighing the substance onto plastic pans and then adding the substance to the dilution water in the mixing vessels. The mixing vessels were sealed with Teflon® covered neoprene stoppers. The mixtures were stirred at <10% vortex for 41 hours and 30 minutes on magnetic stirplates with Teflon® coated stirbars at room temperature. During mixing, the treatments appeared clear / colorless with approximately 95% of the test substance at the surface and approximately 5% of the test substance floating in solution. After mixing, the mixtures were allowed to settle for 4 hours and 5 minutes in a water bath at 20.0C, before the aqueous portions were removed through the outlet at the bottom of the vessels. After settling the mixtures appeared clear / colorless with the test substance on the bottom of the mixing vessel. Four replicates were prepared by filling the test chambers with approximately 140 mL of the WAFs. Four replicates of the control were prepared in the same manner using reconstituted water.

Test Chamber / Volume of Solution - The test chambers were 125 mL size glass Erlenmeyer flasks containing approximately 140 mL of solution (no headspace). The test chambers were closed with ground glass stoppers to minimize contamination, evaporation and/or volatilization.

Exposure Duration - 48 hours (± 1 hour)

Exposure Conditions - Mean test temperature: 19.7°C (S.D. = 0.4).

Diurnal light: approximately 16 hours light and 8 hours dark. The intensity ranged from approximately 646 to 700 Lux during full daylight periods of the study.

Measured daily in the waterbath where the test chambers were located.

Experimental Evaluation - Observations for immobilisation of the daphnids were made on each replicate at 24 and 48 hours (¿ 1 hour). Immobilisation is defined as the lack of swimming ability or movement within 15 seconds after gentle agitation of the test container. In addition, observations for normal or abnormal daphnid behavior or appearance were made.

Discrete Measurements - Water quality measurements (pH, dissolved oxygen and temperature) were performed on a sub-sample of the WAFs from each treatment and control on Day 0 and on a composite of the replicates in each treatment and control at termination. No undissolved test substance was observed in any of the test chambers.

After completion of the study, the test organisms were discarded.

Organism Loading During the Definitive Study - ~ 28 mL of solution per daphnid

Test Acceptability - The test was considered acceptable as none of the control organisms were immobilized or trapped at the surface of the water and the dissolved oxygen levels remained above 60% of the air saturation value at the temperature tested.

Calculations - Binomial theory dictates that when at least 10 organisms are used with zero mortality (immobilisation for Daphnia sp.), there is a 99.9% confidence that the EL50 is greater than the concentration/loading level tested1.


Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 101 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
Binomial theory dictates that when at least 10 organisms are used with zero mortality (immobilisation for Daphnia sp.), there is a 99.9% confidence that the EL50 is greater than the concentration tested. The study resulted in 5% immobilisation at 101 mg/L. It is believed that the EL50 is >100 mg/L since only one out of twenty Daphnia were immobilised at a loading rate of 101 mg/L. The endpoint is based on the actual initial loading level of test substance to dilution medium.

The in-life observation results are summarized below.

 

Loading Level

(mg/L)

24 Hour

(% Survival)

24 Hour

(% Normal)

48 Hour

(% Survival)

48 Hour

(% Normal)

Control

100

100

100

100

101

100

100

95

5*

*Organisms noted as not normal were recorded as lethargic. Lethargy is indicative of a state of lower

   swimming activity compared to the control group.

Validity criteria fulfilled:
yes
Conclusions:
EL50 is >100 mg/L
Executive summary:

This study was performed to evaluate the acute toxicity of a water-accommodated fraction (WAF) of the test substance, to the daphnid,Daphnia magnaStrauss, in a 48 hour static test. This study was performed as a limit test.

The test substance actual loading level for this study was101mg/L. A control treatment consisting of the vehicle/dilution water with no test substance was also prepared. The test substance is a solid. It was ground using a mortar and pestle prior to WAF preparation; none of the particles appeared to be larger than 1 mm from a visual determination. The WAFs were prepared by adding the appropriate amount of test substance to reconstituted water in glass aspirator bottles and stirring on magnetic stirplates for 41 hours and 30 minutes. The mixtures were allowed to settle for approximately 4 hours before removing the aqueous portions (WAFs) for testing.

 

Four replicate chambers per treatment were tested. Each replicate contained five daphnids. Replicate chambers were125mL glass Erlenmeyer flasks containing approximately 140 mL of solution (no headspace). Each chamber was sealed with a ground glass stopper to minimize contamination and evaporation and/or volitilization.Water quality (temperature, pH, and dissolved oxygen) measurements were recorded on Day 0 and at termination. The daphnids were observed for immobilisation and sub-lethal effects at 24 and 48 hours ± 1 hour.

 

Binomial theory dictates that when at least 10 organisms are used with zero mortality (immobilisation forDaphnia sp.), there is a 99.9% confidence that the EL50is greater than the concentration / loading level tested. The study resulted in 5% immobilisation at 101 mg/L. It is believed that the EL50is >100 mg/L since only one out of twentyDaphniawere immobilised at a loading rate of 101 mg/L.

 

Description of key information

Two studies were conducted.

The result of both studies concluded that the Daphnia magna, EL50 (48h) > 100 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 mg/L

Additional information