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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:
2 March 1982 - 6 March 1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline and/or standard method but was non-GLP. There was no analytical confirmation of the test substance.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
analytical characterization was not conducted and mean length of fish were 7.2 cm (6.8-7.5 cm) instead of 5 +/- 1 cm
Principles of method if other than guideline:
not applicable
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:
no data
Vehicle:
yes
Details on test solutions:
0.006 ml/L Emulsogen 8% was used for the highest test concentration

A stock solution was prepared consisting of 1 g TK 10490 blended with 0.613 ml Emulsogen (8%) + 100 ml water, warmed up, treated with ultrasound and completed to 1000 ml with water.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
length: 71.9 mm (68 - 75 mm)
weight: 3.34 g (3.0 - 4.0 g)
loading: 0.445 g/l
acclimatization: 25 days
supplier: P. Hohler, CH-4314 Zeiningen
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
not applicable
Hardness:
191.72 mg/L CaCO3
Test temperature:
15 +/- 2C
pH:
7.9-8.3
Dissolved oxygen:
6.3-9.7 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
nominal- C1 (blank control), C2 (vehicle control), 1, 1.8, 3.2, 5.8, 10 mg/L
Details on test conditions:
Calculated amounts of stock solution to produce the desired concentrations were given into the dechlorinated tap water (hardness 191.72 mg/L CaCO3) of the aquaria and homogenously distributed with a high speed mixer. Ten fish (68-75mm in length weighing 3.0-4.0g) were placed in each glass aquaria, loading of 0.445g/L, with 15L of water and allowed a 48- hour acclimation period. Test concentrations were 1, 1.8, 3.2, 5.8, and 10 mg/L, and fish were fasted 24-hours prior to exposure and throughout the test. A blank control and vehicle control (Emulsogen 8%) were also used.

Water temperature was maintained 15 +/- 2C, and light was provided 16 hours per day. Dissolved oxygen, pH, and temperature were recorded daily. Mortality and behavior were recorded at 24, 48, 72, and 96 hours.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: ; graphically determined
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
1.85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: ; graphically determined
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: ; graphically determined
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: ; graphically determined
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.75 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: ; graphically determined
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: ; graphically determined
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
2.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: -; calculated
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 1.8 - 2.5 mg/l; calculated
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 1.4 - 2.0 mg/L; calculated
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 1.3 - 1.8 mg/L; calculated
Details on results:
LC values were determined by both calculation and graphical analysis.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
see table above

There was no mortality noted in either control, blank or vehicle control, or 1.0 mg/l test material. There was no mortality noted in the first 24 hours at 1.8 mg/l. Within 48, 72 and 96 hr, two, six and eight fish, respectively, had died at 1.8 mg/l. All fish exposed to 3.2, 5.8 or 10 mg/l died within the first 24 hrs.

Swimming behavior, equilibrium, respiratory function and pigmentation were observed in a small number (1-3) of fish exposed to 1 or 1.8 mg/l. All but respiratory function were noted within 24 hours and did not appear to progress. Respiratory function was initially noted in one fish after 24 hr and three fish after 96 hr to 1.8 mg/l.

Acute Toxicity of TK 10 490   
LC50 (96 hour) graphically determined  1.75 mg/L 
LC0 (96 hour) graphically determined  1 mg/L 
 LC100 (96 hour) graphically determined   3 mg/L
  LC50 (96 hour) calculated  1.5 mg/L
 95% confidence limit  1.3 - 1.8 mg/L
 LC50 (72 hours) graphically determined 1.8 mg/L 
 LC50 (72 hours) calculated 1.7 mg/L 
 95% confidence limit  1.4 - 2.0 mg/L
 LC50 (48 hour) graphically determined  1.85 mg/L
 LC50 (48 hour) calculated  2.1 mg/L
 95% confidence limit  1.8 - 2.5 mg/L
 LC50 (24 hour) graphically determined  2.4 mg/L
 LC50 (24 hour) calculated  2.4 mg/L
 95% confidence limit  --
 Controls (blank 96 hour)  0% mortalities
 Controls (vehicle 96 hour)  0% mortalities
Conclusions:
The 96 hour LC50 is 1.5 mg/L in Salmo gairdneri (calculation).
Executive summary:

The LC50 (96 h) in Salmo gairdneri was determined for BADGE. There was no mortality noted in either control, blank or vehicle control, or 1.0 mg/l test material. There was no mortality noted in the first 24 hours at 1.8 mg/l. Within 48, 72 and 96 hr, two, six and eight fish, respectively, had died at 1.8 mg/l. All fish exposed to 3.2, 5.8 or 10 mg/l died within the first 24 hrs. The 96 hour LC50 is 1.5 mg/L in Salmo gairdneri (calculation).

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline and/or standard method but was non-GLP. There was no analytical confirmation of the test substance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
not specified
Analytical monitoring:
no
Details on sampling:
No data
Vehicle:
yes
Details on test solutions:
Three methods were used to prepare stock solutions/dispersions of the test material.
1. Direct addition: measured quantities of the test substance (either by weight or volume) were added directly to the test vessels and dispersed by stirring for a few minutes.
2. Ultrasonic agitation in water: measured quantities of the test substance were added to distilled water and then agitated in a DAWE's Sonicleaner ultrasonic bath for approximately 30 minutes.
3. In acetone: measured quantities of the test substance were added to Analar acetone. Epikote 828 was soluble in acetone over the range of stock solution concentrations used. The concentrations of the stock solutions/dispersions were such that not more than 0.5 ml/L of acetone was added to the test media.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Healthy S. gairdneri fingerlings from two hatcheries were used for the tests; Parkwood Trout Farm, Harrietsham, Kent and Zeals Fish Farm, Wolverton, Wiltshire. The fish were acclimated to the test conditions for more than 10 days before use. The wet weight and fork length of a sample of ten fish from each experiment were determined.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
250-280 mg/L
Test temperature:
14-16 degrees C
pH:
7.7 - 8.4
Dissolved oxygen:
9.0 - 10.7
Salinity:
not applicable
Nominal and measured concentrations:
Nomical concentrations for direct addition: 0 (control), 1.0, 1.5, 2.5, 4.0 mg/L
Nominal concentrations for sonicated addition: 0 (control), 1.0, 2.0, 3.5, 6.0, 10 mg/L
Nominal concentration for solvent (acetone): 0 (control), 1.5, 2.5, 4.0, 6.5, 10 mg/L
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
direct application
Basis for effect:
mortality
Remarks on result:
other: 2.9 - 6.2
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
2.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
acetone solvent carrier
Basis for effect:
mortality
Details on results:
At concentrations greater than 10 mg/L globules of undissolved test substance were observed at the bottom of the test vessels. It is therefore concluded that Epikote 828 was not wholly solube in water at concentrations greater than 10 mg/L under the condition of the tests.

When the test material was sonicated in solution, the resulting LC50 value reported (7.7 mg/L) was considered invalid, since physical effects/toxicity was likely at the dose levels used to determine this LC50 value.
Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test material was determined to be 3.6 mg/L when the material was directly added to the water medium. The 96 hour LC50 for the test material was determined to be 2.3 mg/L when the test material was delivered in acetone.
Executive summary:

Three 96-hour static toxicity tests were carried out with Salmo gairdneri. The test media were renewed daily. The 96 hour LC50 for the test material was determined to be 3.6 mg/L when the material was directly added to the water medium. The 96 hour LC50 for the test material was determined to be 2.3 mg/L when the test material was delivered in acetone. These toxicity values are within the range of the reported water solubility of the test material. When the test material was sonicated in solution, the resulting LC50 value reported (7.7 mg/L) was considered invalid, since physical toxicity was likely to have played a role in the observed toxicity at these dose levels.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline and/or standard method.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
other: EPA-660/3-75-009
Deviations:
no
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Only water soluble fraction of EPIKOTE 828 was tested.
Analytical monitoring:
yes
Details on sampling:
Water samples were taken for analysis at intervals prior to and after the start of the test. During method development work it was shown that the water-soluble fraction of EPIKOTE 828 was composed principally of some species other than the major component of the test material as supplied. Mass spectrometry showed that, though this "water-soluble fraction" used as a standard was not pure, it contained a principal component which was probably a diol formed by hydrolysis of one of the two epoxide moieties present in the major component of EPIKOTE 828.

An analytical standard was obtained by preparative chromatography of EPIKOTE 828 to separate a quantifiable amount of the "water soluble fraction".

Three injections of EPIKOTE 828 in MTBE (3 x 100 mg EPIKOTE) were made on the following HPLC system:
Column: 25 cm x 9 mm I.D. stainless steel
Packing: Partisil 5 PAC
Mobile phase: MTBE + methanol (99.5 + 0.5 v/v)
Flow rate: 5 ml/min
Wavelength: 228 nm
(MTBE - methyl t-butyl ether)

Five fractions were collected following each injection. Fraction 5 co-chromatographed with the contents of the stock solutions and was used to produce analytical standard solutions for use on the following HPLC system:
Column: 25 cm x 4.6 mm I.D. stainless steel
Packing: Spherisorb 5 NH
Mobile phase: MTBE + methanol (92.5 + 7.5 v/v)
Flow rate: 1 ml/min
Wavelength: 228 nm
Retention time: "Water soluble fraction" ca 7.5 -8.2 min.

Vehicle:
no
Details on test solutions:
The "water soluble fraction" obtained from EPIKOTE 828 and used as an analytical standard in the aquatic tests was examined by mass spectrometry. Though it was not possible to characterize this material completely, the results suggested that a major component of the "water soluble fraction" was the diol presumably formed by hydrolysis of one of the two epoxide function of DGEBPA, the major component of EPIKOTE 828
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Healthy O. mykiss fingerlings were obtained from Zeals Trout Farm, Wiltshire and acclimated to the test conditions for more than 9 days before use. A sample of ten of the fish used in the test had a mean length of 3.4 cm (range 3.1 to 3.8 cm) and a mean weight of 0.33 g (range 0.24 to 0.49 g).
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
96 h
Post exposure observation period:
Fish were observed after 96 hours and sacrificed
Hardness:
ranged from 270 to 288 mg/L as CaCO3.
Test temperature:
ranged from 15-16C
pH:
ranged from 7.1 to 8.3
Dissolved oxygen:
ranged from 8.6 to 10.2 mg/L
Salinity:
no data
Nominal and measured concentrations:
Concentration in mg/L with time (T)


Percentage of
saturated solution T0 T24 old T24 new T48 T72 T96 old
0 (Control) <0.010 0.014 <0.010 <0.010
10 0.80 0.76 0.73 0.081
22 1.7 1.6 1.5 1.4
46 3.3 2.7 n/s n/s
100 7.4 4.6 n/s n/s
Details on test conditions:
Ten O. mykiss were placed in each aquarium. The test solutions were gently aerated throughout the test to maintain the concentration of dissolved oxygen. The fish were not fed. At 24 h intervals after the start of the test, the fish were observed and the numbers exhibiting toxic symptoms were recorded. The symptoms were classified into one of five categories from (a) - no toxic symptoms to (e) - dead. After completing these observations, any dead fish were removed, the dissolved oxygen concentration and pH were measured in the control and highest test concentration and the test solutions were renewed. The dissolved oxygen concentration, pH and hardness of the freshly prepared control and highest test concentration solution were also determined prior to introducing fish. The temperature of the test solutions was monitored throughout the test.

Samples of the freshly prepared and 24 h old test and control solutions were analyzed to determine the concentration of the water-soluble fraction of EPIKOTE 828.

Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
4.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: water soluble fraction
Basis for effect:
mortality
Remarks on result:
other: 4.0-5.0
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: water soluble fraction
Basis for effect:
mortality
Remarks on result:
other: 2.2-3.1
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: water soluble fraction
Basis for effect:
mortality
Remarks on result:
other: 1.5-2.1
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: water soluble fraction
Basis for effect:
mortality
Remarks on result:
other: 1.0-1.3
Details on results:
Sublethal toxic effects were recorded in the lowest test concentration of 0.78 mg/L. These included increased cough frequency and hyperventilation.which was observed in 1 of 10 fish after 24 hours, 0 of 10 fish after 48 hours, 4 of 10 after 72 hours and 5 of 10 after 96 hours.
Results with reference substance (positive control):
No positive control study conducted
Reported statistics and error estimates:
See above table

Based upon the results of the test the 24, 48, 72 and 96 h LC50 values, expressed in terms of the concentration of the water-soluble fraction of EPIKOTE 828 present in the test medium were calculated to be 4.4, 2.7, 1.8 and 1.2 mg/L, respectively.

Validity criteria fulfilled:
yes
Conclusions:
Based upon the results of the test the 24, 48, 72 and 96 h LC50 values, expressed in terms of the concentration of the water-soluble fraction of EPIKOTE 828 present in the test medium were calculated to be 4.4, 2.7, 1.8 and 1.2 mg/L, respectively.
Executive summary:

Aqueous stock solutions of the water-soluble fraction of EPIKOTE 828 were generated by recirculating dilution water/culture medium through glass columns packed with inert particulate material (pumice) coated with 5 -10% (m/m) EPIKOTE 828. O. mykiss was exposed to serial dilutions of the stock solutions in tests to determine the acute toxicity of the soluble fraction of EPIKOTE 828 to them.

It should be noted that the water-soluble fraction of EPIKOTE 828 is composed principally of chemical species other than the major component of the test material as supplied. The principal water-soluble component is probably a diol formed by hydrolysis of one of the two epoxide moieties present in the major component of EPIKOTE 828.

In this study the results of the toxicity tests are expressed in terms of the measured concentrations of the water-soluble fraction of EPIKOTE 828 present in the aqueous test media.

The acute toxicity of EPIKOTE 828 to O. mykiss was determined in a 96 h semi-static test with daily renewal of the test media. Based upon the results of the test the 24, 48, 72 and 96 h LC50 values, expressed in terms of the concentration of the water-soluble fraction of EPIKOTE 828 present in the test medium were calculated to be 4.4, 2.7, 1.8 and 1.2 mg/L, respectively.

Description of key information

The 96-hour LC50 value of 2.0 mg/L for BADGE and BADGE-related resins is the geometric mean of four 96-hour LC50 values taken from valid exposure studies with BADGE and rainbow trout (Salmo gairdneri now Oncorhynchus mykiss).

Key value for chemical safety assessment

LC50 for freshwater fish:
2 mg/L

Additional information

Six acute freshwater fish studies were assessed for this endpoint. Four studies were conducted with rainbow trout (Salmo gairdneri, now Oncorhynchus mykiss), one study was conducted with zebrafish (Danio rerio) and one study was conducted with the fathead minnow (Pimephales promelas). The study conducted with fathead minnows was deemed unacceptable for use due to inappropriate temperature for the test species and also due to the fact that the test solutions in the study were above the limit of the solubility. A total of five studies (four with rainbow trout and one with zebrafish) were determined to be of good quality and acceptable for use in risk assessment. The acute tests with BADGE and rainbow trout gave varying 96-hour LC50 values of 1.2, 1.5, 3.6, 2.3 and 74.8 mg/L. The acute test with BADGE and zebrafish reported a 96-hour LC50 of 2.4 mg/L. The four lowest 96-hour LC50 values reported for rainbow trout (1.2, 1.5, 3.6, and 2.3 mg/L) were used to calculate a geometric mean value of 2.0 mg/L for this species. The 96-hour LC50 value of 74.8 mg/L was not used in the calculation of the geometric mean since it was greater than 10-fold different from the other LC50 values. This is in accordance with ECHA guidance from Chapter R.10 (section R.10.2.2). The discrepancy in toxicity values is likely the result of one study testing above the limit of solubility for the test material (however no specific mention of insolubility was made in the study reporting the 96-hour LC50 of 74.8 mg/L). The lowest 96-hour LC50 value of 2.0 mg/L for rainbow trout was selected as the key parameter for this endpoint.