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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:
27 November to 1 December 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, not conducted according to recognised guideline, but fully detailed method and results.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Hardness and alkalinity were determined according to standard analytical procedures (American Public Health Organisation, 1976). Test methodology followed recommended bioassay practices (U.S. Environmental Protection Agency, 1975) with the exception that replicate concentrations were not used.

Fresh stock solution for the test was prepared by weight to a precision of 0,1 mg, diluted with reagent grade acetone and introduced into the test vessels. The test was conducted in 19.6 litre, chemically clean, glass jars containing 15 litres of water. The test was started by introducing -the toxicant into test vessels containing dilution water, thoroughly mixing and then introducing the fish. The amount of solvent in the solvent control equalled the amount used in the highest concentration.

References to books used:
- American Public Health Association. 1976. Standard Methods for the
Examination of Water and Wastewater
- Brauhn, J. L., R. A. Schoettger and L. EI. Mueller. 1975. Acquisition and culture of research fish: rainbow trout, fathead minnows, channel catfish and bluegills
- Committee on Methods forToxicity Tests with Aquatic Organisms. 1975. Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians.
- Finney, D. J. 197 1. Statistical Methods in Biological Assay.
- Marking, L. L . and V. K. Dawson . 1973. Toxicity of Quinaldina Sulfate to Fish. Invest. Fish Control No. 48
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
no
Details on sampling:
Not applicable.
Vehicle:
no
Details on test solutions:
Fresh stock solution for the test was prepared by weight to a precision of 0.1 mg, diluted with reagent grade acetone and introduced into the test vessels. The test was conducted in 19.6 litre, chemically clean, glass jars containing 15 litres of water. The test was started by introducing the toxicant into test vessels containing dilution water, thoroughly mixing and then introducing the fish. The amount of solvent in the solvent control equalled the amount used in the highest concentration.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Strain: Pimephales promelas Rafinesque
- Source: UCES Laboratory
- Age at study initiation: approximately 5 months old
- Length at study initiation (mean) : 51mm
- Weight at study initiation (mean): 1.13g
- Feeding during test: None
- Food type: No data.
- Amount: No data.
- Frequency: No data.

ACCLIMATION
- Acclimation period: 24 hours
- Acclimation conditions: In test water.
- Type and amount of food: No data.
- Feeding frequency: The fish were taken off food 48 hours before the start of the test.
- Health during acclimation (any mortality observed): No data.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
All observations made during exposure period.
Hardness:
Total hardness as CaCO3: 43 mg/l.

Test temperature:
20.8C +/- 0.8C
pH:
7.42
Dissolved oxygen:
The concentration of dissolved oxygen was measured throughout but no mean or required value was given.
Salinity:
Not applicable.
Conductivity:
Not specified
Nominal and measured concentrations:
Five concentrations, a control and solvent control were used in determining the toxicity. Nominal concentrations were: 1.8, 3.2, 5.6, 10.0 and 18.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Jar
- Material, fill volume: Glass, 19.6 litre containing 15 liters of water.
- Aeration: no data.
- No. of organisms per vessel: no data.
- No. of vessels per concentration (replicates): 1 - no replicates
- No. of vessels per control (replicates): 1 - no replicates
- Biomass loading rate: 0.75 g/l.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water obtained from a well on the Tarrytown site, treated
with a Continental Reverse Osmosis Water System (Model 3020) and deionized.
- Alkalinity: 28 mg/L
- Hardness: 43mg/L
- Conductance: 140 pmhos/cm.
- Culture medium different from test medium: no data.

OTHER TEST CONDITIONS
- Adjustment of pH: no data.
- Photoperiod: no data.
- Light intensity: no data.

EFFECT PARAMETERS MEASURED:
- Dissolved oxygen, water bath temperatre and pH were determined initially and every 24 hours afterwards for the control, solvent control and all test concentrations.
- Abnormal behaviour responses of the test fish were noted and recorded at 24 hour intervals.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Various.
- Test concentrations: 1.8, 3.2, 5.6, 10.0 and 18.0 mg/l
Reference substance (positive control):
yes
Remarks:
Sodium lauryl sulfate
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
10.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL
Details on results:
- Behavioural abnormalities: Fathead minnows exposed to concentrations of 5.6 mg/l and higher became hemorrhagic. The fish in the 5.6 mg/l and 18.0 mg/l concentrations also exhibited irritation and sounding. Minnows in the 10.0 mg/1 concentration exhibited abnormal surfacing behavior.
- Observations on body length and weight: No data
- Other biological observations: No data.
- Mortality of control: No.
- Other adverse effects control: None
Results with reference substance (positive control):
- Results with reference substance valid?
- Mortality: Yes
- LC50: 4.2 mg/l after 96 hours (95% confidence interval).

Results:

The 96 hour LC50 with 95% confidence intervals for K-50 to fathead minnows is 10.8 (8.0- 14.6) mg/l .The 96 hour observed no effect level is 3.2 mg/l. It should be noted that LC50 values may vary with different species, temperatures and water qualities.

Table 1: Percent Mortality:

Observation

Control 

Solvent Control 

Test Material, Nominal Concentration (mg/L)

 

 

 

 1.8

 3.2

5.6 

10.0 

18.0 

 24 hour

 0

 48 hour

30 

70 

72 hour 

10 

50 

70 

 96 hour

20 

50 

70 

Table 2: LC50Values:

 

 

24 hour 

48 hour 

72 hour 

96 hour 

LC50 

mg/L 

>18.0 

13.5 

11.4 

10.8 

95% Confidence 

Lower 

N.A. 

10.6 

8.6 

8.0 

Intervals 

Upper 

N.A. 

17.4 

15.2 

14.6 

The 96 hour NOEC (No observed effect concentration) is 3.2 mg/L.

N.A. = not available due to the abscence of sufficient mortalities.

Table 3: Dissolved Oxygen, mg/L:

 

Control 

Solvent Control 

Test 

Material 

Nominal 

Concentration 

mg/L 

 

 

 

1.8 

3.2 

5.6 

10.0 

18.0 

Initial 

9.1 

8.4 

8.5 

8.5 

8.3 

8.3 

8.6 

24 hour 

5.5 

4.5 

3.5 

4.9 

4.0 

4.4 

4.1 

48 hour 

3.2 

1.9 

1.7 

1.8 

1.5 

1.5 

1.5 

72 hour 

3.5 

2.2 

2.1 

2.1 

1.9 

1.8 

2.0 

96 hour 

3.7 

2.4 

2.7 

2.6 

2.3 

2.8 

2.9 

Table 4: pH:

 

Control 

Solvent Control 

Test 

Material 

Nominal 

Concentration 

mg/L 

 

 

 

1.8 

3.2 

5.6 

10.0 

18.0 

Initial 

7.42 

7.42

7.40

7.42 

7.40

7.39

7.39 

24 hour 

7.05

7.04

6.95 

7.08

7.00 

7.03

7.01

48 hour 

6.96

6.86

6.84 

6.88

6.85 

6.85 

6.85 

72 hour 

6.93

6.82

6.84

6.84

6.83

6.86

6.86

96 hour 

6.98

6.88

6.90 

6.90

6.89 

6.94

6.96

Validity criteria fulfilled:
not specified
Conclusions:
This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at > 5%

The LC50 values for the registered substance after 24, 48, 72 and 96 hours are >18.0, 13.5 , 11.4 and 10.8 mg/L respectively. The NOEC vaule was determined to be 3.2 mg/L. Kronitex-50: Synthetic Triaryl Phosphate was therefore determined to be moderately toxic to the fathead minnow, based upon a relative scale for Water Quality Characteristics of Hazardous Materials.
Executive summary:

This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at > 5%

The short-term toxicity of the registered substance to the Fathead minnow (Pimephales promelas Rafinesque) was assessed, with the 96-hour LC50 value determined to be 10.8mg/L and the NOEC value to be 3.2mg/L. Kronitex- 50: Synthetic Triaryl Phosphate was therefore determined to be moderately toxic to the fathead minnow, based upon a relative scale for Water Quality Characteristics of Hazardous Materials.

On the basis of these results, the substance is classified as "harmful" to aquatic organisms.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 May 1997 - 6 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study, conducted to recognised guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
During the fish' holding period, the acclimation temperature of 23 ± 2°C stated in the protocol was exceeded by 0.9°C. The 3. rerio were acclimated to 25.2 to 25.9°C 48 hours prior to test initiation. This deviation did not affect the outcome or validi
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
no
Details on sampling:
Nominal test substance concentrations were used for both the range-finding and definitive tests.
Vehicle:
no
Details on test solutions:
Test solutions of Durad 310M were prepared by weighing out three separate 9.0-g quantities of test substance and adding them directly to 9-L volumes of dilution water, to yield the desired test concentration. A dilution water control was maintained concurrently with the test solutions.

TEST WATER
The dilution water was a moderately hard freshwater. The water originated from the Town of Jupiter and was treated by vigorous aeration, filtered to 20 micrometers, passed through activated carbon and re-aerated prior to use. The dilution water, at test initiation, possessed a hardness of 84 mg/L as calcium carbonate (CaC03) , alkalinity of 15 mg/L as CaCOj, and specific conductivity of 378 micromhos per centimeter (/imhos/cm) .

Chemical characterization of a representative batch of dilution water was recorded.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Juvenile zebra-fish (Brachydanio rerio) used for testing were received on April 2, 1997 at Toxikon Environmental Sciences from Aquatic Research, Hampton, NH. Zebra-fish cultures were maintained in filtered freshwater with a temperature range of 22.0 to 27.2 °C during the 14-day period prior to test initiation. Holding temperature was 25.9 °C when fish were removed for testing. Zebra-fish were maintained on a diet of flake food (Ziegler Bros., Gardners, PA). Zebra-fish appeared to be in good health at test initiation and mortality during the 7 days prior to test initiation was zero percent. No diseases were observed or treated during the holding period. Zebra-fish were fed until 24 hours prior to test initiation, but were not fed after that time or during the test period.

Zebra-fish used for the test ranged from 27 to 30 millimeters (mm) standard length (mean and standard deviation = 29 + 1.1 mm) and from 0.24 to 0.43 grams (g) wet weight (mean and standard deviation = 0.33 ± 0.06 g) as measured from the control fish at test termination. Loading was calculated to be 0.37 g of fish tissue per liter of test solution.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
84 mg/l as CaCO3
Test temperature:
23.0 +/- 0.1 deg C average
pH:
6.8 as ave
Dissolved oxygen:
8.6 mg/l ave
Salinity:
Not applicable.
Conductivity:
Not specified
Nominal and measured concentrations:
Range-finding test was conducted at nominal concentrations of 0.10, 1.00, 10.0, 100, and 1000 mg/L
Main test was conducted at 0 and 1000 mg/L
Details on test conditions:
Methods followed for the 96-hour static test with zebra-fish, Brachydanio rerlo, were those described in Toxikon Environmental Sciences' test protocol entitled: "Durad 310M: Acute Toxicity To Zebra-fish, Brachydanio rerio, Under Static Test Conditions."

A 96-hour static range-finding test was conducted at nominal concentrations of 0.10, 1.00, 10.0, 100, and 1000 mg/L, prior to performing the definitive test. Three fish were tested at each concentration. After 96 hours of exposure, mortality of zebra-fish in the range-finding test was zero percent in all test concentrations and the control. Two fish in the 1000 mg/L test concentration exhibited a complete loss of equilibrium and one fish in the 100 mg/L test concentration exhibited erratic behavior. Based upon the lack of mortality during this preliminary test, a single definitive test concentration of 1000 mg/L was selected to be run in triplicate.

Test solutions of Durad 310M were prepared by weighing out three separate 9.0-g quantities of test substance and adding them directly to 9-L volumes of dilution water, to yield the desired test concentration. A dilution water control was maintained concurrently with the test solutions.

The 96-hour test was initiated on May 2, 1997 with the impartial addition of zebra-fish, by twos, to all test chambers until 10 fish were distributed to each test chamber. The fish were added to the test chambers after the addition of the test substance and the monitoring of initial water quality parameters. The test chambers were 10-L glass jars (22-cm diameter x 30-cm height) containing 9 L of dilution water and providing a water depth of 25 cm. All test chambers were covered throughout the exposure period to reduce evaporation. The test chambers were positioned in a water bath under fluorescent lighting regulated to a photoperiod of 16 hours light and 8 hours darkness. The light intensity ranged between 2.5 and 3.7 microEinsteins per square meter per second as measured by a LI-COR, Inc. Model LI-189 light meter equipped with a 2pi quantum sensor.

Survival of zebra-fish was monitored daily. Any abnormalities in the behavior or physical appearance of the zebra-fish were also noted. Fish were not fed during the test. Aeration of the test solutions began on day 1 (24 hours) of the test to prevent the dissolved oxygen levels from dropping below 60 percent saturation.

The diurnal range of the water bath temperature was continuously monitored using a minimum/maximum thermometer and recorded daily. Water quality (i.e., temperature, pH and dissolved oxygen concentrations) were measured in each test chamber daily. Specific conductivity, total alkalinity, and total hardness of the dilution water were measured at test initiation and termination. Specific conductivity was measured using a Corning Model Checkmate 90 digital conductivity meter. Water hardness and alkalinity were determined by EDTA and potentiometric titration, respectively (APHA et al., 1992). Dissolved oxygen concentrations and chamber temperatures were determined using a YSI Model 58 oxygen meter utilizing a membrane electrode. The pH was measured with a Fisher Scientific Accumet Model 1001 pH meter.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 other: mg wm/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: Based on the test results, a 96-hour LC50 value, 95 percent confidence limits, and slope of the concentration/mortality response curve could not be calculated.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
< 1 000 other: mg wm/L
Nominal / measured:
nominal
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: Based on the test results, a 96-hour LC50 value, 95 percent confidence limits, and slope of the concentration/mortality response curve could not be calculated.
Details on results:
A limit test was performed using a nominal test concentration of 1000 mg wm/L. A film on the test solution surface was observed in all replicates of the 1000 mg wm/L test concentration on days 0 and 1. Undissolved chemical was observed in all 1000 mg wm/L replicates throughout the entire test. Furthermore, the 1000 mg wm/L test solutions were observed to be cloudy on days 3 and 4.

Mortality of the zebra-fish exposed for 96 hours to Durad 310M was zero percent in the single test concentration of 1000 mg wm/L and in the dilution water control (Table 1). The 96-hour LC50 was >1000 mg/L and could not be calculated due to the lack of mortality during this test. The no-observed-effect-concentration (NOEC) was <1000 mg/L based on observed sublethal effects at 1000 mg wm/L. Sublethal responses are described in Table 1 below.

The test temperature ranged from 22.0 to 23.5°C during the test. Initial dissolved oxygen concentrations in all control and test chambers were 8.4 to 3.5 mg/L (98 to 99% of saturation) and remained between 5.7 mg/L and 9.0 rag/L (67 to 105% of saturation) for the remainder of the exposure (Table 4). Aeration was initiated to all control and test chambers at 24 hours of exposure to insure dissolved oxygen concentrations remained >60% of saturation; the dissolved oxygen concentrations had dropped to 5.7 - 6.5 mg/L (67 to 76% of saturation at 24 hours). The pH of all test solutions ranged between 6.4 and 7.0 throughout the test, while the pH of the dilution water control was 7.4 at test initiation and ranged from 6.8 to 7.4 during the remainder of the test.
Results with reference substance (positive control):
None
Reported statistics and error estimates:
Based on the test results, a 96-hour LC50 value, 95 percent confidence limits, and slope of the concentration/mortality response curve could not be calculated.

Table 1.   Mortality of Zebra-fish,Brachydanio rerio,During a 96-Hour Static Exposure to Durad 310M

 

Nominal

Concentration (mg/L)             

 

Cumulative Number Dead  (Percent Mortality)

 

(mg/L)

 

24 Hour

48 Hour

72 Hour

96 Hour

Control

A

0

(0)

0

(0)

0

(0)

0

(0)

 

B

0

(0)

0

(0)

0

(0)

0

(0)

 

C

0

(0)

0

(0)

0

(0)

0

(0)

1000h

A

0

(0)

0

(0) (a,j)

0

(0) (c,j)

0

(0) (f,j)

 

B

0

(0)

0

(0) (j)

0

(0) (d,j)

0

(0) (d,j)

 

C

0

(0)

0

(0) (b,j)

0

(0) (c,j)

0

(0) (e,g,j)

 

a) Three fish with dark coloration and ten fish were slightly less active in comparison to control fish.

 

b) Three fish exhibited complete loss of equilibrium;  three fish were gyrating; and one fish had dark coloration. 

 

c) Three fish exhibited complete loss of equilibrium; three fish had dark coloration.

 

d) One fish exhibited complete loss of equilibrium; three fish exhibited erratic behavior.

 

e) Five fish exhibited complete loss of equilibrium; six fish had dark coloration.

f) Five fish exhibited complete loss of equilibrium; five fish had dark coloration.

 

g) Three fish exhibited erratic behavior.

 

h) Undissolved chemical was observed thoughout the test and film on the surface of test solution was observed on days 0 and 1.

 

i) Gentle aeration was initiated in all chambers due to dissolved oxygen levels approaching 60 percent saturation.

 

j) Test solution was cloudy.

Validity criteria fulfilled:
yes
Conclusions:
This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at < 5%

A static acute toxicity test of Durad 310M (lot 96Y-101) was conducted in the zebra fish. Brachydanio rerio, according to OECD Guidelines for Testing of Chemicals, Method 203. Based on preliminary exposures, a limit test was performed. Three groups of ten B. rerio were exposed to a nominal concentration of 1000 mg/L of the test material in water. A similarly comprised unexposed control group was included. Observations for mortality and sublethal effects were made daily. Water quality parameters such as pH, temperature and dissolved oxygen were recorded at prescribed intervals.
The exposure concentration clearly exceeded the solubility limits of the test material. A surface film and undissolved test material were observed during the test. Exposure to this supersaturated solution of Durad 310 M produced no mortality among the exposed fish. Sublethal effects included dark coloration, loss of equilibrium and erratic behavior. Gentle aeration was required after 24 hours to maintain the dissolved oxygen concentrations above 60% of saturation
Executive summary:

This test was conducted on a sample of the material where the content of Triphenyl phosphate is present at < 5%

 

A static acute toxicity test of Durad 310M (lot 96Y-101) was conducted in the zebra fish.Brachydanio rerio,according to OECD Guidelines for Testing of Chemicals, Method 203. Based on preliminary exposures, a limit test was performed. Three groups of tenB. reriowere exposed to a nominal concentration of 1000 mg/L of the test material in water. A similarly comprised unexposed control group was included. Observations for mortality and sublethal effects were made daily. Water quality parameters such as pH, temperature and dissolved oxygen were recorded at prescribed intervals.

The exposure concentration clearly exceeded the solubility limits of the test material. A surface film and undissolved test material were observed during the test. Exposure to this supersaturated solution of Durad 310 M produced no mortality among the exposed fish. Sublethal effects included dark coloration, loss of equilibrium and erratic behavior. Gentle aeration was required after 24 hours to maintain the dissolved oxygen concentrations above 60% of saturation. The substance is not considered to be harmful at the levels of concentration tested in the study.

Description of key information

Short-term toxicity to fish

Key value for chemical safety assessment

LC50 for freshwater fish:
10.8 mg/L

Additional information

Studies are presented for a variety of species as follows:

Where TPP content > 5%

 

Pimephales promelas: LC50 (96- h):10.8 mg/L test mat. (nominal)(K2)

Salmo gairdneri (new name: Oncorhynchus mykiss):LC50 (96- h):1.6 mg/Lmg/L test mat. (nominal)(K2)

Pimephales promelas:LC50 (96- h): 50.1 mg/L test mat. (nominal) (K2)

Salmo gairdneri (new name: Oncorhynchus mykiss):LC50 (96- h):4.46 mg/Lmg/L test mat. (nominal)(K2)

 

Where TPP content < 5%

 

Brachydanio rerio (new name: Danio rerio):LC50 (96 h): > 1000 mg wm/L dissolved (nominal) (K1)

The data set available for the phosphates as a group indicates that these do pose hazardous effects to fish.  However, such effects are considered to be attributable to the content of the impurity, triphenyl phosphate, CAS 115-86-6, (EC No. 204-112-2).  The content of this impurity is considered to adversely affect the toxicity of the substance within aquatic organisms. The registration dossier that two separate classifications applicable to the substance for this endpoint as follows:

• Phenol, isopropylated, phosphate (3:1) [Triphenyl phosphate >5%] - H410: Very toxic to aquatic life with long lasting effects.

• Phenol, isopropylated, phosphate (3:1) [Triphenyl phosphate < 5%] - H413: May cause long lasting harmful effects to aquatic life.

For the purposes of hazard classification, the following studies are taken as key studies:

Where TPP content > 5%

Pimephales promelas: LC50 (96- h):10.8 mg/L test mat. (nominal) (K2)

 

Where TPP content < 5%

Brachydanio rerio (new name: Danio rerio):LC50 (96 h): > 1000 mg wm/L dissolved (nominal) (K1)