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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9th to 13th December 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Principles of method if other than guideline:
Protocol Deviations:
Due to the size of available test organisms, the loading rate (0.8 g/L) was greater than 0.5 g/L. Test results were not presented graphically because there was 97% survival at the single tested concentration. These deviations did not adversely affect the outcome of the test and no other deviations were made from the study protocol.

Protocol Amendments:
The protocol was amended to specify that the nominal concentrations for the definitive test would be 0 and 1,000 mg/L and that each concentration would be replicated three times.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
WAFs were prepared at the beginning of the test and three additional times during the test to allow media renewal at 24, 48, and 72 hours. At 0 hour, two WAFs were prepared at 1,000 mg/L by combining 30.0 g of test substance and dilution water in a glass mixing vessel equipped with a magnetic stirrer and adjusting the final water volume to 30 liters. The 24, 48, and 72 hour WAFs were prepared by combining 25.0 g of test substance and dilution water and adjusting the final water volume to 25 liters. The vortex of the WAF was approximately 10%. The mixtures were stirred for approximately 20 hours and allowed to settle for approximately 4 hours. Following the settling period the water phase containing the WAF was removed from the mixing vessel with a siphon and the two WAFs were combined.

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Test Organisms: Juvenile trout were obtained from Mount Lassen Trout Farms, Red Bluff, CA. At the end of the experiment the fish had an average
wet weight of 1.2 g and an average length of 49.9 mm. The loading rate for the test was 0.80 g/L. Fish were held for 14 days prior to use in the test,
and in the last two days prior to testing there was less than 3% mortality. Fish were fed during the holding period except for the 48 hours prior to use Fish were not fed during the test.
TEST ORGANISM
- Common name: rainbow trout
- Source: Mt. Lassen Trout Farms, Red Bluff, California
- Age at study initiation (mean and range, SD): Juvenile
- Feeding during test: Fish were provided with dry commercial food (Tetra-min Lot # TMO6) each day during acclimation except during the 48 hours before the start of the test. Fish were not fed during the test.


ACCLIMATION
- Acclimation period: During the 14 day period before the start of the test, the temperature range was 12.1 to 13.0°C and the dissolved oxygen concentration was always at least 8.8 mg/L.
- Acclimation conditions (same as test or not): Prior to testing the fish were maintained under flow-through conditions in a 270 liter fiberglass tank.
- Health during acclimation (any mortality observed): During acclimation fish were not treated for disease and they were free of apparent disease, injuries, and abnormalities at the beginning of the test and there was less than 3% mortality during the 48 hours prior to the test.

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
Dilution Water: Deionized water was collected at Marblehead, MA and adjusted to a hardness of 40-48 mg/L as CaCO3. The water was continuously
aerated and passed through a particle filter, ultraviolet sterilizer and activated carbon during storage in a polyethylene tank. Water used for the test
had a hardness of 44 mg/L as CaCO3 and an alkalinity of 33 mg/L as CaCO3.
Test temperature:
11.8 to 12.8 deg C.
pH:
7.0 – 7.6
Dissolved oxygen:
7.1 – 10.2 mg/L
Nominal and measured concentrations:
Control and 1,000 mg/L WAF loading rate.
Details on test conditions:
Test System: Treatment levels were control and 1000 mg/L loading rate. Individual water accommodated fractions (WAFs) were prepared for each
renewal of the 1000 mg/L treatment. Renewals occurred at 24-hour intervals. A measured weight of test material was added to a measured volume
of dilution water (25 or 30-L) in a glass vessel and stirred for 20 hours. Stirring was accomplished using a magnetic stir bar. Mixing speed was
adjusted such that a vortex formed that reached approximately 10% of the distance to the bottom. Following the mixing period, the test solutions
were allowed to stand for 4 hours before the water phase was siphoned off. The siphoned water phase (i.e., WAF) was used in the aquatic toxicity test.
The control and 1000 mg/L treatments consisted of three replicates each of 15 L test solution in 20L aquaria which were lightly covered. Ten fish
were used per replicate (thirty per treatment).

Light: 16-h light per day using cool-white fluorescent lights with an intensity of approximately 4 µEin/m2sec.

Water conductivity: 130 – 170 µmhos/cm.
TEST SYSTEM
- Test vessel: 20 liter glass aquaria
- Type (delete if not applicable): loosely covered with plastic
- Material, size, headspace, fill volume: containing 15 liters of test solution. At the end of the test, water depth was approximately 18 cm.
- Aeration: Aeration was not required to maintain dissolved oxygen concentrations above acceptable levels.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates):
- Biomass loading rate: 0.8 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Water used for acclimation of test organisms and for all toxicity testing was deionized water collected at Marblehead, Massachusetts. Water was adjusted to a hardness of 40 to 48 mg/L as CaCO3 and stored in 500-gallon polyethylene tanks where it was aerated and continuously passed through a particle filter, ultraviolet sterilizer, and activated carbon. Dilution water used for the definitive test had a hardness of 44 mg/L and an alkalinity of 33 mg/L as CaCO3.
- Conductivity: 130 – 170 umhos/cm.


OTHER TEST CONDITIONS
- Photoperiod: A 16 hour light and 8 hour dark photoperiod was automatically maintained with cool-white fluorescent lights
- Light intensity: 4 µEin/m2sec.


TEST CONCENTRATIONS
- Range finding study
A range-finding test was conducted under static conditions from November 14 to 18, 1996 with a dilution water control and the WAF of two concentrations of OLOA 216Q, 100 and 1,000 mg/L. The range-finding test was conducted with a single replicate of each concentration, each containing ten organisms. At the end of the 96 hour exposure period there was 0% survival in the control, 90% survival at 100 mg/L, and 100% survival at 1,000 mg/L OLOA 216Q. This test was invalid due to poor control survival, but the data was used to estimate the test substance concentration to be used in the definitive toxicity test.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Observations on body length and weight: At the conclusion of the test the control fish had an average wet weight of 1.2 g (the loading rate was 0.80 g/L) and an average total length of 49.9 mm.
- Mortality of control: One hundred percent survival occurred in the control
Reported statistics and error estimates:
Statistical analysis of the survival data could not be conducted because greater than 50% survival occurred at the tested concentration. The NOEC is the highest concentration that did not cause toxicant related effects (survival was at least 90% and no sublethal effects were noted).
Results of the toxicity tests could not be interpreted by standard statistical techniques (Stephan, 1983) because there was at least 90% survival in all test vessels. The no observed effect concentration (NOEC) is the highest tested concentration that allowed at least 90% survival of exposed organisms and did not cause sublethal effects.
Sublethal observations / clinical signs:

Nominal concentrations: 24, 48, 72 and 96-h LL50 (reported as “LC50” in the report) >1,000 mg/L. This is equivalent to 96-h LL0 (reported as “NOEC” in the report) = 1,000 mg/L.

Measured concentration: n/a

Analytical Monitoring: TOC levels at 0 hours were between 1.7 and 2.4 mg/L for the control and 2.0 and 3.6 mg/L for

the 1,000 mg/L treatment. TOC levels at 24 hours were between 2.1 and 4.1 mg/L for the control and 2.4 and 3.7 mg/L for the 1,000 mg/L treatment. The active ingredient is in oil and TOC levels are not considered to be indicative of actual test material concentrations. Results are therefore based on nominal loading rates.

Test Findings: Control survival was 100%, and 1000 mg/L treatment survival was 97%. No insoluble test material was observed in the test vessels during the entire aquatic toxicity test.

Test results reported in original study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates. Control response was satisfactory.

No significant mortality or signs of toxicity were observed in the 1000 mg/L WAF loading rate treatment or in the control throughout the entire test.

Validity criteria fulfilled:
yes
Conclusions:
The 24, 48, 72, and 96 hour LL50s were each greater than 1,000 mg/L (expressed as the nominal amount of test substance used to prepare the WAF), and the 96 hour NOELR was 1,000 mg/L.
Executive summary:

The acute toxicity of the water accommodated fraction (WAF) of a 1,000 mg/L mixture of the test material and water to the rainbow trout, Oncorhynchus mykiss, was investigated during a study conducted at T.R. Wilbury Laboratories, Inc. under GLP conditions and in accordance with OECD guideline 203. The test, which was designed to determine the toxicity of the WAF of the test substance, was performed from December 9 to 13, 1996.

The test was performed at 12 ± 1°C under static, renewal conditions with a control and the WAF of a 1,000 mg/L mixture of test substance and water. The dilution water was carbon filtered deionized water collected at Marblehead, Massachusetts and adjusted to a hardness of 44 mg/L as CaCO3. Juvenile rainbow trout used for the test were procured from a commercial supplier and acclimated to test conditions for at least 14 days prior to use in the test.

The WAF was prepared by formulating a 1,000 mg/L solution of the test material in dilution water and placing it in a glass mixing vessel equipped with a magnetic stirrer. The mixture was stirred for approximately 20 hours, settled for approximately 4 hours, and the water phase containing the WAF was siphoned off. Approximately 80% of the media was renewed in each test vessel after 24, 48, and 72 hours using a freshly prepared WAF. There was no insoluble material observed during the test.

The WAF of the test substance was not toxic to rainbow trout, Oncorhynchus mykiss, at the tested concentration. The 96 hour median lethal concentration (LC50) of the test material to rainbow trout was greater than 1,000 mg/L (expressed as the nominal amount of test substance used to prepare the WAF) and the no observed effect concentration (NOEC) was 1,000 mg/L.

Nominal concentrations: 24, 48, 72 and 96-h LL50 (reported as “LC50” in the report) >1,000 mg/L. This is equivalent to 96-h NOELR (reported as “NOEC” in the report) = 1,000 mg/L.

Test results reported in the study as “lethal concentrations” are reported in this summary as “lethal loading”, because test results are based on WAF loading rates. Control response was satisfactory.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14th to 18th April 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study following GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
minor deviations considered not to affect the outcome of the study
Principles of method if other than guideline:
Guideline Deviations:
- The protocol states that fish used will be at 2.0 ± 1.0 cm total length. For this study fish that were 9 - 29 mm total length were used. As this was range was close to the specified length, and the average length was 1.9 cm, it is the opinion of the testing laboratory that this deviation did not affect the results of this study.
- The protocol states that the total mortality of the test fish population during the 7 days prior to testing will be less than 5%. For this test mortality data was available for only six days prior to testing. As the fish were of good health, and the population had no mortality during this period, it is the opinion of the testing laboratory that this deviation did not affect the results of this study.

GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
not specified
Details on test solutions:
DILUTION WATER: Natural filtered seawater from Cape Cod Canal, Bourne, Massachusetts

DESCRIPTION OF 100% WSF: a clear colorless solution , containing a black viscous liquid which adhered to the sides and bottom of the test vessel.
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
The sheepshead minnows, (Bionomics lot # 86A24) were obtained from a commercial supplier in Massachusetts and held in a glass aquaria or fiberglass holding tank under a photoperiod of 16 hours light and 8 hours darkness. A closed loop recirculating filtration system provided natural filtered (5 um) seawater with a salinity range of 32 - 34 ‰, a pH range of 7.6, and a dissolved oxygen concentration range of 92 - 93 % of saturation (Weekly Record of Fish Holding Water Characteristics). Test fish were maintained under these conditions for a minimum of six days. The temperature range in the holding tank was 19 - 21 °C during this period. The fish were fed <48 hours old (post hydration) Artemia nauplii, ad libitum, daily until the day prior to testing. There was no mortality of the test fish population during the six days prior to testing (Daily Record of Fish Holding Conditions). The mean wet weight of the test fish population was 0.17 g (range 0.030 - 0.44 g, N=30) and the mean total length was 19 mm (range 9 - 29 mm, N=30) (Fish Weight and Length Log). Test fish were 33 days old at the time of test initiation.
Test type:
semi-static
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
21 - 23 deg C
pH:
7.6-8.2
Dissolved oxygen:
Dissolved Oxygen: 6.5-6.9 mg/L (new), 5.4-6.2 (aged solutions)
Salinity:
32 ‰
Nominal and measured concentrations:
0 and 10,000 mg/L
Details on test conditions:
No range finding study was conducted.

A measured weight (150 g) of test material was added to a measured volume of water (15 L) in a glass vessel and stirred for 16 to 20 hours. Stirring was accomplished using a shaker table. Following 2 hours of settling, duplicate 20 mL samples were removed for total carbon analysis.
The water-soluble fraction (WSF) was separated from floating or settled material by gently pumping 5 L of WSF from a point approximately midway between the bottom, surface and sides of the jar into replica test aquaria. Duplicate control aquaria were also established. Greater than 90% of the test
solution was replaced daily with freshly prepared test solution. The test solutions were maintained at 22 deg C. Test solutions were not aerated.
Fish were not fed during exposure.

All organisms were observed for mortality and the number of individuals exhibiting clinical signs of toxicity or abnormal behavior at 0, 24, 48, 72
and 96 hours after initiation of test material exposure.

The toxicity test was conducted in 18.9-L glass aquaria which contained 5.0 L of test solution. The test solution depth was 6.0 cm with a surface area of 985 cm2. All test solution temperatures were controlled by a water bath system designed to maintain temperatures at 22 ± 1°C. Test solutions were not aerated. The photoperiod during testing was the same as that provided in the fish culture area. Illumination was provided at an intensity of 25 foot candles with a Color Rendering Index >91 by a combination of Sylvania Growlux and Cool White fluorescent bulbs.

Ten sheepshead minnows, selected impartially from the holding tank, were placed in each test aquaria within 10 minutes after the test solutions had been transferred. The resulting test organism loading concentration was 0.080 grams of biomass per liter of test solution. Fish were not fed during exposure.
Reference substance (positive control):
yes
Remarks:
sodium lauryl sulfate
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 10 g/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
A sodium lauryl sulfate (SLS) reference test was conducted with the test fish population from 21 - 25 April 1986. The resulting 96 hour LC50 was 1.3 mg/L with a 95% confidence interval of 0.72 - 2.0 mg/L .
Reported statistics and error estimates:
No data
Sublethal observations / clinical signs:

The cumulative percent mortalities and the observations made during the preliminary Tier I test with 100% WSF (10 g/L) are presented in Table 1. Based on these results the 100% WSF (10 g/L) of the test material is not toxic to ≥ 50% of the test organism population and was therefore not subject to Tier II definitive testing. The pH, dissolved oxygen concentrations, salinity and temperatures measured during the toxicity test are presented in Table 2.

Validity criteria fulfilled:
yes
Conclusions:
The 96-hour mortality of the fish tested at 100% Water Soluble Fraction (WSF) was 5 %. Based on this result, no Tier II toxicity test was necessary.
The LL50 can therefore be consired to be >10 g/l.
Executive summary:

In an acute toxicity study conducted in accordance with GLP to the methodologies set out in OECD guideline 203, the 96-hour mortality of the fish tested at 100% Water Soluble Fraction (WSF) was 5% Based on this result, no Tier II toxicity test was necessary. The LL50 can therefore be consired to be >10 g/l.

The test was conducted according to the Protocol for Static Renewal Acute Toxicity Test with Cyprinodon variegatus Following OECD Guidelines, February 1986, and Protocol Amendments Dated 18 February 1986.

Description of key information

 There is sufficient acute aquatic toxicity to fish data available for the registered substance, including in a marine species. All studies available resulted in no to minimal acute aquatic toxicity.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
1 000 mg/L

Marine water fish

Marine water fish
Effect concentration:
10 000 mg/L

Additional information

In the key study (Ward, 1997), the acute toxicity of the water accommodated fraction (WAF) of a 1,000 mg/L mixture of the test material and water to the rainbow trout, Oncorhynchus mykiss, was investigated during a study conducted at T.R. Wilbury Laboratories, Inc. The test, which was designed to determine the toxicity of the WAF of the test substance, was performed from December 9 to 13, 1996 and was conducted according to the OECD Guideline 203 (Fish, Acute Toxicity Test) and GLP.

This study is presented as the key information as the reliability rating for this study is 1, according to the criteria of Klimisch, 1997, and this study was conducted most recently and was therefore chosen as the most reliable study.

There are two supporting studies available:

- The T.J. Ward, 1993 study (T.R. Wilbury Laboratories, Inc., report number: 9176-CMA/ESI) was not considered the key study as it was conducted less recently than the above key study. The study was conducted according to the OECD Guideline 203 (Fish, Acute Toxicity Test) and GLP and was considered to have a reliability rating of 1, according to the criteria of Klimisch, 1997.

Nominal concentrations: 96-h LL50 (reported as “LC50” in the report) >1,000 mg/L. This is equivalent to 96-h LL0 (reported as “NOEC” in the report) of 1,000 mg/L.

Test results reported in the study as “lethal concentrations” are reported in this summary as “lethal loading rate”, because test results are based on WAF loading rates.

- Supporting information, obtained from the 2000 European Chemicals Bureau IUCLID Data Set for the substance is presented, however this is secondary source information and is considered to have a reliability rating of 4, according to the criteria of Klimisch, 1997.

Marine conditions:

- The Nicholson R.B. and Breteler R.J. 1986 study (Springborn Bionomics, Inc. report number: BW-86-04-1981) is considered the key study for acute toxicity to marine fish. The study was conducted according to the OECD Guideline 203 (Fish, Acute Toxicity Test) and GLP and was considered to have a reliability rating of 1, according to the criteria of Klimisch, 1997.

The 96-hour mortality of the fish tested at 100% Water Soluble Fraction (WSF) was 5% Based on this result, no Tier II toxicity test was necessary. The LC50 can therefore be consired to be >10 g/l.