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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
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 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No data
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):
Daphnia magna
Details on test organisms:
Daphnia magna, less than 24-hr old, were taken from a culture in STL, derived form a strain obtained from I.R.C.H.A., France.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
220 mg/L
Test temperature:
19-21 degrees C
pH:
7.8 - 8.3
Dissolved oxygen:
8.6 - 9.2 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations for direct application: 0 (control), 1.0, 2.0, 5.0, 10, 20, and 50 mg/L
Nomical concentrations for sonicated application: 0 (control), 1.0, 2.0, 5.0, 10, 20, and 50 mg/L
Nominal concentrations for acetone solvent application: 0 (control), 0.2, 0.5, 1.0, 2.0, 5.0, 10, and 20 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
2.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Direct addition
Basis for effect:
mobility
Remarks on result:
other: 2.3 - 3.5
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
Sonicated
Basis for effect:
mobility
Remarks on result:
other: 0.81 - 1.4
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.4
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
carried in acetone solvent
Basis for effect:
mobility
Remarks on result:
other: 1.2 - 1.6
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.
Validity criteria fulfilled:
not specified
Conclusions:
The EC50 for Daphnia magna ranged from 1.1 - 2.8 based on varying methods of test material application.
Executive summary:

Three 48 hour static toxicity tests were conducted with Daphnia magna. The test material was delivered by either direct additon, with sonication, and with an acetone carrier solvent. The resulting EC50 values (based on immobility) were determied to be in the range of 1.1 - 2.8 mg/L, which was within the range of water solubility for the test material.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Sept 1984 - 14 Oct 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP; however, there was no analytical confirmation of the test substance solutions reported in this study.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Analytical monitoring:
no
Details on sampling:
No analytical monitoriing was conducted.
Vehicle:
yes
Details on test solutions:
The test solutions were prepared by adding small quantities of stock solutions of Epikote 828 in acetone to 100 ml of water in the dishes. The total amount of acetone added to each dish, including the controls, was adjusted to 0.01 ml (0.1 ml/L)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: D. magna
- Strain/clone: The culture was derived froma clone held at ICI Brixham Laboratory, supplied originally to ICI by IRChA, France.
- The D. magna used in the test were less than 24 hours old.

The D. magna are held in 0.5 to 4 liters of water in suitably sized glass vessels, at densities of up to 20 animals per liter. They are kept in a temperature controlled room (20 plus or minus 2 degrees C) under artificial cool white light regulated to give a light/dark cycle of 16h/8h. The cultures are changed to fresh water at least once per week. This is a reconstituted hard water (pH 7.0 - 8.5; hardness 160 to 220 mg/L as CaCO3), identical to that used for the toxicity tests. The cultures are fed daily with Chlorella vulgaris and active dried yeast. Typical feeding rates are 10^6 cells/mL of Chlorella and 5 mg/L of yeast. The cultures are sieved regularly to remove young. Adults are first removed temprarily by pipette and the culture vessel contents passed through a sieve (5.6 mm mesh) on which the young animals are retained. The water is returned, with the adult D. magna to the culture vessel.

Young for use in toxicity tests are less than 24 hours old and are colleted from cultures ages between 15 and 35 days. 24 h before a test is set up the culture is sieved and the young discarded. The culture is then re-sieved 23 h later. D. magna retained on the sieve are then left in clean water for 1 h (they are now up to 24 hours old) and those swiming actively are used for testing. Young for testing are not taken from cultures which contain ephippia; these cultures are discarded.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no data
Hardness:
160 mg/L CaCO3 (initial)
Test temperature:
18-22 degrees C
pH:
8.1-8.3
Dissolved oxygen:
9.1-9.2 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations: 0, 0.1, 0.2, 0.5, 1, 2, 5, 10
Details on test conditions:
A 48 hour static toxicity test was carried out without renewal of the test solutions. Test vessels were made in a series of concentrations ranging from 0.1-10 mg/L with reconstituted fresh water. D. magna (less than 24 hours old) were placed in 150 ml glass dishes containing 100 ml of test solution, with 10 animals per dish and 3 replicate dishes per treatment. After 24 and 48 hours, the numbers of immobilized daphnia were recorded. They were considered immobilized if, after stirring the flask, they did not swim during a 10 second observation period. The temperature of the test solutions was 18-22C, pH ranged from 8.1-8.3, the total water hardness was 160 mg/L as CaCO3, and the concentration of dissolved O2 was 9.1-9.2 mg/L.
Reference substance (positive control):
not specified
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 3.8 - 5.6
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
4.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 1.4 - 2.1
Details on results:
At 48 hours, all of the D. magna had died at 3 mg/L while none had died at 1 mg/L.
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
see above

At 48 hours, all of the D. magna had died at 3 mg/L while none had died at 1 mg/L. The 48 h EC50 value from the acute toxicity test was 1.7 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
The 48 hour EC50 is 1.7 mg/L.
Executive summary:

A 48 hour static toxicity test was carried out with Daphnia magna. Test vessels were made in a series of concentrations ranging from 0.1-10 mg/L with reconstituted fresh water. D. magna (less than 24 hours old) were placed in 150 ml glass dishes containing 100 ml of test solution, with 10 animals per dish and 3 replicate dishes per treatment. After 24 and 48 hours, the numbers of immobilized daphnia were recorded. The 24 hour EC50 was 4.6 mg/L and the 48 hour EC50 was 1.7 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not stated
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline
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):
not relevant
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):
Daphnia magna
Details on test organisms:
Daphnia magna, less than 24 h old, were taken from a laboratory culture. The culture is derived from a strain obtained (via ICI Brixham Laboratory) from I.R.Ch.A., France.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Animals were sacrificed at the end of the study.
Hardness:
164 - 170 mg/L as CaCO3
Test temperature:
19 - 20
pH:
8.3 - 8.4
Dissolved oxygen:
9.6 mg/L
Salinity:
not relevant
Nominal and measured concentrations:
Concentration (mg/L)
% saturated dose T0 T48
0 (reconstituted fresh water) <0.010 <0.010
0 (medium from Chromosorb <0.010 <0.010
control column
2.2 0.23 0.27
4.6 0.60 0.71
10 1.0 1.2
22 2.2 2.6
46 4.8 5.5
100 10 11
Percent of saturated solution
A recovery experiment was conducted at a fortification rate of 0.077 mg/L and indicated the rate of recovery of the water soluble fraction of EPIKOTE 828 from water to be 100%.
Details on test conditions:
A 48 h static toxicity test was carried out without renewal of the test solutions. A saturated stock solution of the water-soluble fraction of EPIKOTE 828 in reconstituted fresh water was produced. Eight, duplicate sets of 150 ml conical flasks were prepared. To five pairs of these were added quantities of the stock solution and reconstituted fresh water sufficient to produce, when completely full, 46, 22, 10 4.6 and 2.2% dilutions of the stock solution. Two of the other three pairs of flasks were filled either with the undiluted stock solution or reconstituted fresh water - the latter to serve as a control. The final pair of flasks was filled with reconstituted fresh water that had been circulated through a column packed with Chromosorb WHP that had not been coated with EPIKOTE 828. The latter were also to serve as controls and are subsequently referred to as chromosorb controls.

Ten D. magna, less than 24 h old, were allocated to each flask and the flasks sealed with a screw cap. After 24 and 48 h the numbers of imrnobilised D. magna in each flask were determined and recorded. D. magna were considered to be immobile if, when the contents of the flask were briefly swirled they did not swim during a 15 second period of observation.

The pH and dissolved oxygen concentration in the control and highest concentration test solutions were determined at the start and conclusion of the test. The total hardness of the water used in the preparation of the control and test solutions was determined at the start of the test. The temperature of the control and test solutions was monitored throughout the test.

Samples of the freshly prepared and 48 h old test and control solutions were analysed to determine the concentration of the water-soluble fraction of EPIKOTE 828.


Reference substance (positive control):
not specified
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
4.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
other: water soluble fraction
Basis for effect:
mortality
Remarks on result:
other: 3.6 - 7.2
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: 1.6 - 4.1
Details on results:
The highest concentration in which no immobilization of D magna was recorded in the test was 1.1 mg/l.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
see above

The 24- and 48 -hour LC50 was 4.9 and 2.7 mg/L, respectively.

Conclusions:
The 24- and 48 -hour LC50 was 4.9 and 2.7 mg/L, respectively.
Executive summary:

The acute toxicity of EPIKOTE 828 to D. magna was determined in a 48 h static test i.e. without renewal of the test media. Based upon the results of the test the 24 and 48 h EC50 values, expressed in terms of the concentration of the water-soluble fraction of EPIKOTE 828 present in the test medium, were calculated to be 4.9 and 2.7 mg/L, respectively.

Description of key information

The 48-hour EC50 value of 1.8 mg/L for BADGE and BADGE-related resins is the geometric mean of five 48-hour EC50 values taken from valid exposure studies with BADGE and Daphnia magna.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.8 mg/L

Additional information

Six acute toxicity studies with Daphnia magna were assessed for this endpoint, and five of these studies were found to be of good quality and reliable for use in the risk assessment process. One study was deemed not reliable (Klimisch score = 3) due to insufficient data and the fact that the test material was reported to have come out of solution and was not wholly soluble at the concentrations tested. Of the five studies that were deemed of good quality and reliable for use in risk assessment (Klimisch score = 1 or 2), one study was only 24 hours in duration, and was therefore classified as a supporting study, and not a key study. Three studies reported 48-hour EC50 values ranging from 1.1 to 2.8 mg/L and one study reported a 48-hour EC50 value of 51.8 mg/L BADGE. Since one EC50 value was significantly greater than the other values, this data point was not factored into the calculation of a geometric mean for determining the 48-hour EC50 value for D. magna. This was in accordance with ECHA guidance (R.10.2.2) which states that if toxicity values are more than one order of magnitude different for the same species, it is questionable whether the data point should be incorporated together into the geometric mean calculation for that species and endpoint. In this case, although not reported in the study, it is probable that the test material was added to the test vessels at concentrations well above its water solubility, therefore the EC50 value of 51.8 mg/L BADGE was not incorporated into the geometric mean calculation. Thus, the key parameter for the freshwater invertebrate studies was the EC50 value of 1.8 mg/L, which was the geometric mean of five 48-hour EC50 values (1.1, 1.4, 1.7, 2.7, and 2.8 mg/L) reported in three studies with D. magna.