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Ecotoxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: ASTM E729-80
Deviations:
not applicable
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples were taken and analyzed for selected organic compounds, pesticides, and PCBs.
Vehicle:
no
Details on test solutions:
Dilution water was pumped from the upper Saginaw Bay of Lake Huron off Whitestone Point. The dilution water was then limed and flocculated with ferric chloride at the Midland water treatment plant. Additionally, the water was carbon filtered, UV irradiated and pH adjusted with CO2 before being used in the lab. Water was monitored for pH, conductivity, hardness, and alkalinity on a weekly basis.
Test organisms (species):
Pimephales promelas
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
The test organisms were not fed 48 hrs prior to exposure.
Hardness:
Hardness ranged from 83 to 94 mg/L as CaCO3
Test temperature:
The temperature in which the minnows were raised was held at 17+/-1 °C.
pH:
The pH ranged from 7.9 to 8.1
Conductivity:
conductivity ranged from 120 to 170 umhos/cm
Nominal and measured concentrations:
nominal Bisphenol A concentrations of 8.65, 5.62, 3.65, 2.37, 1.54, and 1.00 mg/L in addition to a dilution water control
Details on test conditions:
Alkalinity ranged from 49 to 60 mg/L as CaCO3
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
2.28 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
mortality (fish)
Remarks on result:
other: flow-through
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.6 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
mortality (fish)
Remarks on result:
other: flow-through
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.7 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
mortality (fish)
Remarks on result:
other: static
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
2.26 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
mortality (fish)
Remarks on result:
other: static
Sublethal observations / clinical signs:

Preliminary Static Acute Test: Nominal Test Concentrations: 0 (control), 1.00, 1.54, 2.37, 3.64, 5.6, 8.2, and 13.3 mg/L Analyzed Test Concentrations: ND, 0.99, 1.43, 2.26, 3.97, 5.51, 8.54, and 14.54 mg/L. 24 to 96 hour LC50 value was 4.7 mg/L, (4.0-5.5, 95 % confidence interval). After 24 hrs, 100 % mortality (10 of 10 test organisms) occurred at analyzed test concentrations of 14.54, 8.54, and 5.51 mg/L. No mortality occurred at exposure concentrations of 3.97 mg/L but at 24 and 48 hrs all fish were lethargic and melanized. At 72 and 96 hours most fish appeared to recover with the exception of one fish that was still lethargic, melanized, and exhibited a loss of equilibrium. At Bisphenol A concentrations of 2.26, 1.43, 0.99 mg/L and the control, no mortality or adverse effects were noted. The LC50 value was calculated using nonlinear interpolation and confidence intervals were obtained by a binomial test. Control fish were measured and weighed at the end of the test and resulted in a mean length of 1.96 cm (range 1.4-2.4 cm) and mean weight of 0.103 g. The loading factor was 0.103 gm fish per liter. Water temperature in the acute test ranged from 17.0 to 17.5 °C, pH from 7.0 to 8.1, and the lowest DO was 65 % of saturation at 17 °C. Flow-Through Test: Nominal Test Concentrations: 0 (control), 1.00, 1.54, 2.32, 3.65, 5.62, and 8.65 mg/L Analyzed Test Concentration: ND, 0.79, 1.52, 2.28, 3.58, 5.4, and 8.66 mg/L. 24 hr LC50 value for flow-through conditions using the mean analyzed concentrations was 4.7 mg/L with a 95 % confidence interval of 3.6-5.4 mg/L. For 48 to 96 hrs the LC50 value was 4.6 mg/L with a 95 % confidence interval of 3.6 -5.4 mg/L. At a Bisphenol A test concentration of 8.66 mg/L, 100 % mortality (20 of 20 test organisms) was observed after 24 hrs. In minnows exposed to a concentration of 5.41 mg/L, 85 % mortality (17 of 20 organisms) occurred after 24 hrs and 90 % was observed after 48 hrs (18 of 20). No additional deaths were recorded after 48 hrs. In the surviving fish, a loss of equilibrium was observed at 24 hrs with one recovering by the end of the study. No deaths occurred in organisms exposed to concentrations of 3.58, 2.28, 1.52, 0.79, or the control group, although in the group exposed to 3.58 mg/L, 9 fish exhibited a loss of equilibrium after 24 hrs, 4 fish at 48 hrs, 2 fish at 72 hrs, and all fish appeared normal at 96 hrs. The fish used in the control group were measured and weighed at the end of the study. The mean standard length of these control fish were 3.0 cm (range 2.5 -4.0 cm) and the mean weight was 0.41 grams. In this test, a fish loading factor of 0.043 g fish per liter per day occurred. Water temperature in the flow-through test ranged from 16.1 to 17.9 °C. The pH ranged from 7.6 to 8.0 and the lowest DO value was 81 % of saturation at 17 °C.

Conclusions:
Static and flow-through acute toxicity tests showed essentially the same 96 hr LC50 values and confidence intervals, 4.7 (4.0-5.5) and 4.6 (3.6-5.4) mg/L respectively. No toxic effects were observed at or below the analyzed Bisphenol A concentrations of 2.26 and 2.28 mg/L in static and flow-through tests respectively. These results indicate that static acute toxicity tests are effective in characterizing the toxicity of a test material that is stable in the test system.
Executive summary:

The acute freshwater toxicity to the fathead minnow, Pimephales promelas, was determined in both static and flow-through exposures according to ASTM E729-80 guidance. This is similar to the OECD 203 guideline. The results from the static and the flow-through exposures were very similar and demostrated that Bisphenol A was stable in the test system during the course of the study. The 96-hr static acute LC50 value (and 95 % confidence interval) was determined to be 4.7 (4.0 -5.5) mg/L. The 96-hr flow-through acute LC50 value (and 95 % confidence interval) was determined to be 4.6 (3.6 -5.4) mg/L. No effects were observed at or below the analysed Bisphenol A concentrations of 2.26 and 2.28 mg/L in static and flow-through tests, respectively.

Endpoint:
short-term toxicity to fish
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 203 (Fish, Acute Toxicity Test)
Deviations:
no
Principles of method if other than guideline:
Method also adheres to OPPTS Draft Guideline 850.1075
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the 96-hour definitive study, samples were removed from one replicate solution of each treatment level and the control at test initiation and test termination. Samples were removed from alternate (i.e., A and B) replicates at each sampling interval and were collected from the approximate midpoint of the test vessel using a pipet.
Vehicle:
no
Details on test solutions:
Based on preliminary testing conducted at Springborn Smithers, nominal bisphenol A test concentrations of 3.1, 6.3, 13, 25 and 50 mg/L were selected for definitive testing. Approximately 10.0 g of the test substance (9.96 g when adjusted for purity of the test substance) was brought to a volume of 1.5 L with deionized water. Each solution was sonicated and stirred with a water-driven stir plate for approximately one hour at 40 ºC. The pH of each stock was adjusted to approximately 11.3 by the addition of 10.5 to 11.0 mL of 10 N NaOH. Each stock was brought to a final volume of 200 L with 20 ppt filtered seawater and observed to be clear and colorless with no visible undissolved test substance.
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
Juvenile sheepshead minnow (Cyprinodon variegatus) was selected as the test organism. The sheepshead minnow used during this study (SSL Lot No. 09A23) were obtained from Aquatic BioSystems, a commercial supplier located in Fort Collins, Colorado. Prior to testing, the fish were held in a 500-L fiberglass tank with a closed-loop recirculating filtration system under a photoperiod of 16 hours light and 8 hours darkness. The seawater which flowed into this tank had a salinity of 21‰. Test fish were maintained under these conditions for a minimum of 14 days. The temperature in the holding tank was maintained at 23 ºC during this period. The fish were fed Ziegler Brothers Prime Flakes, ad libitum, daily.
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
temperature range of 22 to 23 ºC
pH:
pH: 7.7 to 7.9
Salinity:
Salinity: 20 to 21‰
Nominal and measured concentrations:
Nominal concentrations 3.1, 6.3, 13, 25 and 50 mg/L. Mean measured concentrations 3.1, 6.3, 13, 25 and 50 mg/L.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
11 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Remarks on result:
other: 8.3 mg/L to 26 mg/L
Details on results:
All water quality parameters measured were unaffected by the concentrations of bisphenol A tested and remained within acceptable ranges for the survival of sheepshead minnow. Daily measurement of the temperature in the test solutions and continuous temperature monitoring established that the exposure solution temperature ranged from 22 to 23 ºC during the definitive study. Mean measured concentrations ranged from 81 to 130% of nominal and defined the treatment levels as 2.5, 8.3, 12, 26 and 53 mg/L. Following 96 hours of exposure, 70, 100 and 100% mortality was observed among fish exposed to the 12, 26 and 53 mg/L treatment levels, respectively. Two of the surviving fish exposed to the 12 mg/L treatment were observed to have a complete loss of equilibrium and one was observed to be on the bottom of the tank. No mortality or adverse effects were observed among fish exposed to the remaining treatment levels tested (2.5 and 8.3 mg/L) or the control. The 96-hour LC50 value was estimated by binomial probability to be 11 mg/L, with 95% confidence intervals of 8.3 to 26 mg/L. The No-Observed-Effect Concentration (NOEC) was determined to be 8.3 mg/L.
Reported statistics and error estimates:
The 96-hour LC50 value was estimated by binomial probability to be 11 mg/L, with 95% confidence intervals of 8.3 to 26 mg/L.
Validity criteria fulfilled:
yes
Conclusions:
The 96-hour LC50 value was estimated by binomial probability to be 11 mg/L, with 95% confidence intervals of 8.3 to 26 mg/L. The No-Observed-Effect Concentration (NOEC) was determined to be 8.3 mg/L.
Executive summary:

The purpose of this study was to determine the toxicity of Bisphenol A based on the results (LC50) of a 96-hour flow-through acute toxicity test using a representative salt water fish species, the sheepshead minnow (Cyprinodon variegatus). The methods employed in the study meet the testing requirements of the Ecological Effects Test Guidelines draft OPPTS 850.1075 Fish Acute Toxicity Test, Freshwater and Marine, OPPTS 850.1000 Special Considerations for Conducting Aquatic Laboratory Studies, and OECD Guideline for Testing of Chemicals #203, Fish Acute Toxicity Test. The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter and a set of 12 exposure vessels with 10 fish per tank, 20 per concentration. Based on preliminary testing, nominal Bisphenol A test concentrations of 3.1, 6.3, 13, 25 and 50 mg/L were selected for definitive testing. Mean measured concentrations ranged from 81 to 130% of nominal and defined the treatment levels as 2.5, 8.3, 12, 26 and 53 mg/L. Following 96 hours of exposure, 70, 100 and 100% mortality was observed among fish exposed to the 12, 26 and 53 mg/L treatment levels, respectively. The 96-hour LC50 value was estimated by binomial probability to be 11 mg/L, with 95% confidence intervals of 8.3 to 26 mg/L. The No-Observed-Effect Concentration (NOEC) was determined to be 8.3 mg/L.

Endpoint:
short-term toxicity to fish
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:
other: ASTM E729-80
Principles of method if other than guideline:
Method in line with ASTM E729-80 and EPA 600/3-75-009
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Test organisms (species):
Menidia menidia
Test type:
flow-through
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
pH:
6.7-7.7
Dissolved oxygen:
87-95 % saturation
Salinity:
20-33 o/oo
Conductivity:
22,000 and 40,000 umhos/cm (specific conductance)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
9.4 mg/L
Nominal / measured:
meas. (not specified)
Basis for effect:
mortality (fish)
Sublethal observations / clinical signs:

Nominal Concentrations of 5.4, 8.2, 13, 20, and 30 mg/L, correspond to measured concentrations of 4.8, 5.9, 9.8, 16, and 26 mg/L, respectively. QA Testing: Recovery efficiency from the blind samples ranged from 95 to 110 %. Variability between treatment replicates was less than 6 %. LC50s: 24-hr, 12 mg/L (9.8-16 mg/L, 95 % Confidence Interval); 48-hr, 11 mg/L (5.9-16 mg/L, 95 % CI); 72-hr, 9.4 mg/L (8.3-11 mg/L, 95 % CI); 96-hr, 9.4 mg/L (8.3-11 mg/L, 95 % CI). No Discernible Effect Concentration: <4.8 mg/L 100 % mortality (20 out of 20) occurred in Atlantic silversides exposed to Bisphenol A concentrations of 16 and 26 mg/L after just 24-hrs. Exposure to 9.8 mg/L resulted in no mortalities after 24-hrs, but all fish were lethargic, one exhibited a partial loss of equilibrium, and several had a complete loss of equilibrium. After 48-hrs 35 % mortality (7 out of 20) was reported, with all remaining fish observed as being lethargic and one showing signs of a partial loss of equilibrium. After 72-hrs, 50 % mortality(10 out of 20) had occurred, with several of the remaining fish exhibiting a darkened pigmentation and all were reported to be lethargic. The same conditions were reported at 96-hrs as they were at 72-hrs. Atlantic silverside test organisms exposed to a Bisphenol A concentration of 5.9 mg/L had a 0 % mortality throughout the entire exposure period of 96-hrs. Although no mortality occurred, several fish were lethargic or exhibited a partial loss of equilibrium after 24 and 48-hrs. After 72-hrs several fish had a darkened pigmentation and at the end of the study (96 hrs) several were lethargic and showed darkened pigmentation. In organisms exposed to the lowest Bisphenol A concentration of 4.8 mg/L, no mortality occurred until 72 -hrs of exposure when 1 fish died, (5 %). No adverse biological or sublethal effects were recorded at this test concentration. The control treatment resulted in no mortality or adverse effects throughout the exposure period. LC50 values for 24 and 48 -hr exposure were calculated using nonlinear interpolation and the 95 % confidence interval by binomial probability. Probit analysis was used to calculate the LC50 value and 95 % confidence interval for 72 and 96 -hr exposure periods. The pH ranged from 7.9 -8.3 during the 96-hr study period.

Validity criteria fulfilled:
yes
Conclusions:
The 96-hr LC50 value was calculated using probit analysis as 9.4 mg/L. Over the same period of time, a concentration of less than 5.9 mg/L was determined to have "no discernible effect" on the fish. At 4.8 mg/L, a single mortality (5 %) occurred at 72-hrs, however, besides this statistically insignificant death, no mortality was recorded at the next higher concentration. The lack of mortality seen at the next higher dose suggests that 5.9 mg/L could be considered the NOEC.
Executive summary:

A 96 -h flow-through acute study exposed the saltwater Atlantic silverside, Menidia menidia, to Bisphenol A at measured concentrations ranging from 4.8 to 26 mg/L. The 96-hr LC50 value was calculated using probit analysis as 9.4 mg/L. Over the same period of time, a concentration of less than 5.9 mg/L was determined to have "no discernible effect" on the fish. A single mortality (5 %) occurred at 72-hrs at 4.8 mg/L, however, the lack of mortality at the next highest dose suggests that 5.9 mg/L could be considered the NOEC.

Description of key information

There are three key studies which report of acute toxicity to fish for fresh or marine water. The lowest LC50 value in each compartment was used in the risk assessment to provide a conservative assessment. Alexander et al., 1985, (published in Alexander et al., 1988) determined an LC50 of 4.6 mg/L in an ASTM E729-80 study with Pimephales promelas (fresh water). Springborn Bionomics, 1985a (published in Alexander et al., 1988) reported an LC50 of 9.4 mg/L in an OECD 203 study with Menidia menidia (marine water).


 


 

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
4.6 mg/L

Marine water fish

Marine water fish
Dose descriptor:
LC50
Effect concentration:
9.4 mg/L

Additional information

The Klimisch 1 studies are supported by studies with minor restrictions which are scored Klimisch 2:

Bayer, 1998, conducted a study with Danio rerio according to OECD 204 and reported a NOEC of 3.2 mg/L. Emmitte, 1978, followed no guideline in a study with Cyprinodon variegatus and reported a LC50 of 7.5 mg/L. Reiff & Phil, 1979, assessed Onchorhynchus mykiss in a study following OECD 203 and reported an LC50 in the range of 3-5 mg/L. Finally, Bayer, 1989, performed a study with Danio rerio according to a test method proposed by the German Federal Environment Agency and reported a LC50 of 9.9 mg/L.

This set of supporting studies report of LC50 (NOEC in one case) in the range of 3 - 9.9 mg/L.In contrast, other acute fish studies which are listed and discussed in this chapter were rated as Klimisch 3 (not reliable) due to major short-comings or Klimisch 4 (not assignable) due to e.g. insufficient documentation, and  were therefore disregarded in the risk assessment and CLP classification (MITI, 1992, Den Haan, 1992, Kashiwada, 2002).

In conclusion, there are three Klimisch 1 key studies, one for the freshwater compartment (Alexander 1985) reporting a LC50 of 4.6 mg/L and two marine water studies reporting LC50 of 9.4 and 11 mg/L (Springborn Bionomics, 1985, and Sayers, 2009). These studies were used for the risk assessment and CLP classification.