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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
Study period:
29 April 2003 to 27 August 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the final test duplicate samples for analysis were taken from all test solutions and the blank-control.
Sampling:
Frequency: at t=Oh and t=48h.
Volume: 5 ml from the approximate centre of the test vessel.
Storage: Not applicable, the samples were analysed on the day of sampling.
Vehicle:
no
Details on test solutions:
The standard test procedures required generation of test solutions which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. film of the test substance on the water surface).
The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of 97.3% and the substance was not completely soluble in test medium at the concentration tested. The water solubility of Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0x1 0E-4 g/l, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, was determined to be ≥ 5.1*10E6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).
All solutions at loading rates of 1.0 mg/l and higher were prepared separately. Following a short period of ultrasonification (in the final test only), these supersaturated solutions were magnetically stirred for two days to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a floating layer and test substance particles. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter (ca. >.5 µm). The filtrate was clear and colourless. Note that test solutions in the limit test originated from the simultaneously performed acute toxicity study in carp (NOTOX Project 364995).
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th. brood, showing no signs of stress such as mortality > 20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnia with an age of < 24 hours, from parental daphnids of more than two weeks old.

BREEDING
Start of each batch: With new-born animals, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all-glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.- P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period specified.
Hardness:
The hardness: 250 mg/l expressed as CaC03 and the pH: 8.0 ± 0.2 after aeration.
Test temperature:
The temperature of the test medium was 21.6°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.2 and 21.2°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
pH:
7.6 - 7.8
Dissolved oxygen:
8.3 - 8.9
Salinity:
Not applicable - freshwater test
Nominal and measured concentrations:
Measured concentrations
Details on test conditions:
TEST CONCENTRATIONS
Range: Based on the results of the limit test, filtrates (ca. 5 µm) prepared at loading rates of 1.0, 10 and 100 mg/l, and a 10-fold dilution of the filtrate prepared at 1.0 mg/l.
Controls: Test medium without test substance or other additives (0 mg/I).

TEST PROCEDURE AND CONDITIONS
Test type: Static
Test duration: 48 hours
Test vessels: 100 ml, all-glass
Medium: ISO. prepared in mill-RO water
Number of daphnia: 20 per concentration
Loading: 5 per vessel containing 80 ml medium
Light: 16 hours photoperiod daily
Feeding: No feeding
Aeration: No aeration of the test solutions.
Introduction of daphnia: Within % hours after preparation of the test solutions.

MEASUREMENTS AND RECORDINGS
Immobility: (Including mortality) At 24 hours and 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control(s).
Temperature of medium: Continuously in a temperature-control vessel, beginning at the start of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate.
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 1.1 - 1.4 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
LIMIT TEST
Measured concentrations: The test substance consisted of a mixture of molecules with different molecular weights, which differed in water solubility, resulting in a number of peaks in the chromatogram of the test substance solutions. It was not possible to determine which molecule was responsible for the toxicological response, if any. Furthermore, since not all components were quantifiable in the calibration chromatograms, it was not possible to determine the concentration of the total test substance. Therefore, the toxicological evaluation was based on the water-soluble fraction at the loading rate. In addition, the actual concentration was estimated from the three largest peaks (peaks 1, 2 and 3) observed in the chromatograms of HATCOL 3331.
The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l contained an initial concentration of 0.50 mg/l (based on peaks 1 and 2) or 0.48 mg/l (based on peak 3). At the end of the test period the test concentration had decreased below the limit of detection (based on all peaks).
The average exposure concentration, based on peak 1, 2 or 3, respectively, was 0.22, 0.22 or 0.22 mg/l. Hence, the average concentration was above the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l). The observed decrease was probably a consequence of this extremely low solubility.

Immobility: After 48 hours of exposure, 9 out of 20 daphnids exposed to the filtrate had become immobilised. From 24 hours onwards, a floating layer was observed in the filtrate. The EC50 was expected to approximate the average exposure concentration of 0.22 mg/l.
Based on the results of the limit test, the ECso was expected to approximate the water solubility of HATCOL 3331. The actual concentrations were below 1.0 mg/l, and in fact only slightly higher than the limit of quantification. Therefore, the performance of a full study with filtrates prepared at loading rates increasing step-wise by a factor less than 10 would not add to an accurate determination of an EC50. Following from this, a final study was designed using loading rates spaced by a factor of 10.

FINAL TEST
Measured concentrations: Initial concentrations in the filtrates prepared at 1.0, 10 and 100 mg/l were respectively < 0.1, 2.0 and 1.7 mg/l (based on peak 1), < 0.1, 1.9 and 1.6 (based on peak 2) or < 0.1, 1.9 and 1.6 (based on peak 3). After 48 hours, the concentration in the 1.0 mg/l filtrate had remained below the limit of quantification (for all peaks), while the concentrations in the 10 and 100 mg/l filtrate had decreased respectively to 0.83 and 0.72 mg/l (based on peak 1), 0.96 and 0.88 mg/l (based on peak 2) or 1.1 and 0.99 mg/l (based on peak 3).
The average exposure concentration in the 1.0 mg/l filtrate approximated the water solubility (i.e. < 0.1 mg/l), while average exposure concentrations in the 10 and 100 mg/l filtrates were maintained above the water solubility of HATCOL 3331. This was confirmed by the presence of a floating layer in these solutions (10 and 100 mg/l filtrates).

Immobility: The responses recorded in this test were unexpected considering the results of the limit test. Although the concentration in this test were higher than in the limit test, no immobilisation was observed. Similarly to the limit test, a floating layer was observed in the filtrate prepared at 100 mg/l, as well as in the filtrate prepared at 10 mg/l. At the end of the test, the daphnia exposed to these filtrates were examined under a dissection microscope, and found to be covered by test substance. Such observation was not included in the limit test. Even though test substance was attached to the daphnia, this did not seem to hamper them in their movements and mobility. The reason for the immobility in the limit test remains unclear. However, the results of the final test, in which 2 concentrations tested were above the water solubility limit and 1 concentration approximated this limit, while no significant immobility was observed, are considered sufficient to conclude that the EC50 for immobilisation is above the water solubility of HATCOL 3331. Note that a response of 10% is accepted for the controls and therefore not considered to be related to treatment.

Experimental conditions: These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: > 7 mg/l at the start, ≥ 5 mg/l at the end of the test).
The temperature of the test medium was 21.6°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.2 and 21.2°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).

ACCEPTABILITY OF THE TEST
1. In the control, no daphnia became immobilised or trapped at the surface of the water.
2. The oxygen concentration was > 5 mg/l at the end of the test. Other test conditions (pH, temperature) remained within the limits described by the protocol.
3. The 24h-EC50 (based on initial concentrations) of potassium dichromate was within the range of 0.6 to 1.7 mg/l.
Results with reference substance (positive control):
48-hour Acute Toxicity Study in Daphnia magna with K2Cr2O7 (NOTOX Project 381882).
The study procedures described in this report were based on the ISO International Standard 6341, the EEC directive 92/69, Part C.2. "Acute toxicity for Daphnia", December 1992, and the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984.
The reference test was carried out to check the sensitivity of the test system as used by NOTOX. Daphnia were exposed for a maximum of 48 hours to K2Cr2O7 concentrations of 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/l and to a blank control. Ten daphnia were exposed per concentration.
The reference substance, potassium dichromate (K2Cr2O7 art. 4864, batch no. K28974764) was obtained from Merck, Darmstadt, Germany.
The actual responses in this reference test with KzCrz07 are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at NOTOX.
The 24h-EC50 was 1.2 mg/l with a 95% confidence interval between 1.0 and 1.5 mg/l.
The 48h-EC50 was 0.60 mg/l with a 95% confidence interval between 0.52 and 0.76 mg/l.
The raw data from this study are kept in the NOTOX archives. The test described above was performed under GLP.
Reported statistics and error estimates:
Not specified in the study report.

Table 1: Incidence of immobility in the limit test:

Loading rate HATCOL 3331 (mg/l)

Vessel number

Number Daphnia exposed

Response at 24h1

Response at 48h

Number

%

Number

%

Blank-control

A

5

0

0

0

0

 

B

5

0

 

0

 

 

C

5

0

 

0

 

 

D

5

0

 

0

 

100

A

5

1 (2)

5

3

45

 

B

5

0

 

2

 

 

C

5

0 (2)

 

1

 

 

D

5

0

 

3

 

1Between brackets: number of daphnids trapped at the surface. These organisms were reimmersed into the solution before recording of mobility.

 

Table 2: Average exposure concentrations during the final test

Loading rate HATCOL 3331 (mg/l)

Concentration, mg/l

Based on peak 1

Based on peak 2

Based on peak 3

t=0

t=48

Average

t=0

t=48

Average

t=0

t=48

Average

0.10

0.0501

n.d.

n.a.

0.0501

n.d.

n.a.

0.0501

n.d.

n.a.

1.0

0.0501

0.0501

0.050

0.0501

0.0501

0.050

0.0501

0.051

0.050

10

1.97

0.83

1.3

1.94

0.96

1.4

1.90

1.05

1.4

100

1.66

0.72

1.1

1.62

0.88

1.2

1.60

0.99

1.3

n.d., not detected; n.a., not applicable

1measured concentration was below limit of quantification; therefore set at LOQ/2, i.e. 0.050 mg/l

 

Table 3: Acute immobilisation of daphnia after 24 and 48 hours in the final EC50 test

Loading rate HATCOL 3331 (mg/l)

Average concentration (mg/l)

Vessel number

Number Daphnia exposed

Response at 24h1

Response at 48 h1

Number

%

Number

%

Blank-control

-

A

5

0 (1)

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

0.10

-

A

5

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0 (1)

0

1.0

< 0.1

A

5

0

0

0

0

B

5

0 (2)

0

C

5

0

0

D

5

0

0

102

1.3-1.4

A

5

0 (5)

0

03

0

B

5

0 (5)

03

C

5

0 (5)

0 (1)3

D

5

0 (5)

03

1002

1.1-1.3

A

5

0 (5)

0

03

5

B

5

0 (5)

0 (1)3

C

5

0 (5)

13

D

5

0 (5)

03

1between brackets: number of daphnia trapped at the surface. These organisms were reimmersed into the respective solutions before recording of mobility.

2a floating layer was observed starting at 24 h

3all daphnids were observed to be covered with test substance.

 

Table 4: pH and oxygen concentrations during the final test

Loading rate HATCOL 3331 (mg/l)

Average concentrations (mg/l)

Start (t=0 h)

End (t=48 h)

pH

O2

pH

O2

Blank-control

-

7.7

8.3

7.7

8.9

0.10

-

7.7

8.3

7.8

8.8

1.0

< 0.1

7.7

8.3

7.8

8.8

10

1.3-1.4

7.6

8.3

7.8

8.8

100

1.1-1.3

7.6

8.4

7.7

8.7

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:

Concentration (mg/l)

Number Exposed

% immobile

Expected response (%) After 48 hours1

24 h

48 h

Minimal

Maximal

Blank-control

10

0

0

0

102

0.10

10

0

0

0

10

0.18

10

0

0

0

10

0.32

10

0

03

0

30

0.56

10

0

30

0

100

1.0

10

10

100

40

100

1.8

10

100

100

100

100

1Based on historical data of the previous years (n>60)

2A maximum response of 10% does not invalidate the results of the test

3One daphnia was observed trapped at the surface of the test solutions. This daphnid was reimmersed in the respective solutions before scoring of mobility.

Validity criteria fulfilled:
yes
Conclusions:
Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for daphnia could not be reached. Therefore, the 48h-EC50 exceeded the maximum solubility of HATCOL 3331 in test medium.
Executive summary:

Acute Toxicity Study in Daphnia magna with HATCOL 3331.

The study procedures described in this report were based on the ISO International Standard6341: 'Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus-Acute toxicity test, Third edition, 1996-04-01. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2. "Acute Toxicity for Daphnia", and the OECD guideline No. 202 Part I: "Daphnia sp., Acute Immobilisation Test", Adopted April 4, 1984.

The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of 97.3% and the substance was not completely soluble in test medium at the concentration tested. The watersolubilityof Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0x10E-4 g/l, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, wasdetermined to be ≥ 5.1*10E6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).

All solutions at loading rates of 1.0 mg/l and higher were prepared separately. These supersaturated solutions were magnetically stirred for two days to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a floating layer and test substance particles. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter (ca. > 5 µm). The filtrate was clear and colourless.

The project was started with a limit test, exposing daphnia to a filrate (ca. > 5 µm) prepared at a loading rate of 100 mg/l and a blank-control. The test was performed in duplicate with 5 daphnids per vessel and samples for analysis were taken at the start and the end of the test. The analytical results showed that the average exposure concentration was higher than the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l). At the end of the test, 45% of the daphnids exposed to the filtrate were immobilized. Since concentrations were all below 1.0 mg/l, a final test was performed using a range of concentrations nominally spaced by a factor of 10.

A final test was performed exposing daphnia to filtrates (ca. > 5 µm) prepared at loading rates of 1.0, 10 and 100 mg/l and a 10-fold dilution of the filtrate at 1.0 mg/l. Samples for analysis taken at the start and the end of the test showed that the average exposure concentration in the 1.0 mg/l filtrate approximated the water solubility (i.e. < 0.2 mg/l), while average exposure concentrations in the 10 and 100 mg/l filtrates were maintained above the water solubility of HATCOL 3331.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

HATCOL 3331 did not induce acute immobilisation of Daphnia magna at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration above the water solubility, i.e. 1.1 -1.4 mg/l after 48 hours of exposure (NOEC). Also, no immobilisation was observed at concentrations approximating the water solubility.

In conclusion: Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for daphnia could not be reached. Therefore, the 48h-EC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 March 2003 to 26 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the limit test duplicate samples were taken from the blank-control and the filtered solution prepared at a loading rate of 100 mg/l for analysis. The method of analysis is described in the appended Analytical Report.
Sampling:
Frequency: At t=0 and t=48h
Volume: 10 ml from the approximate centre of the test vessels.
Storage: Not applicable, samples were analysed on the day of sampling.
Additionally, the filter used preparation of the test concentration was stored in a freezer for possible analysis in case no test concentrations could be analysed in the samples taken at the start of the test.
Vehicle:
no
Details on test solutions:
The standard test procedures required generation of test solutions that contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions.
The testing of concentrations that disturbed the test system were prevented (e.g. film of the test substance on the water surface).
HATCOL 5236 is a clear colourless liquid with a purity of 96.7%. The water solubility of HATCOL 5236 at 20.0 ± 0.8°C was determined to be < 2.0x10e-4 g/l using the flask method (NOTOX Project 365041).
A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution was stirred for two days to reach maximum solubility. After the stirring period the mixture was hazy and contained a test substance floating layer. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filer (Schleicher and Schuell 604) to remove the larger undissolved test substance particles (ca. > 5µm). The filtrate was still slightly hazy.
Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.
Source: In-house laboratory culture with a known history.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%. presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
Characteristics: For the test selection of young daphnia with an age of <24 hours, from parental daphnids of more than two weeks old.

BREEDING
Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 10 litres of medium in an all glass culture vessel.
Maximum age of the cultures: 4 weeks
Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
Temperature of medium: 18-22°C
Feeding: Daily, a suspension of fresh water algae.
Medium: M7, as prescribed by Dr. Elendt-Schneider.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
No post exposure observation period specified.
Hardness:
The hardness: 250 mg/l expressed as CaCO3 and the pH: 8.0 ± 0.2 after aeration.
Test temperature:
The temperature of the test medium was 20.8°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.0 and 21.3°C during the test.
pH:
pH: 6.0-8.5, not varying by more than 1 unit
Dissolved oxygen:
oxygen: >7 mg/l at the start, ≥5 mg/l at the end of the test
Salinity:
Not applicable - freshwater study
Nominal and measured concentrations:
Measured concentrations
The test substance is a substituted polymer, resulting in a number of peaks in the chromatogram of the test substance solutions. Quantification was based on the two largest peaks. The analytical results showed that the filtered solution prepared at a loading rate of 100 mg/l had an initial concentration of 5.6 mg/l (when based on peak 1) or 5.2 mg/l (when based on peak 2). After 48 hours of exposure the test concentration had decreased to 0.97 mg/l (when based on peak 1) or 0.75 mg/l (when based on peak 2). Hence, concentrations remained above the solubility limit of HATCOL 5236 (i.e. < 0.2 mg/l throughout the test. The observed decrease was probably a consequence of this extremely low solubility.
The average exposure concentration. based on peak 1, was 2.3 mg/l, while the average exposure concentration, based on peak 2, was 2.0 mg/l.
Details on test conditions:
TEST CONCENTRATIONS
HATCOL 5236: A 5 µm filtrate of a supersaturated solution prepared at a loading rate of 100 mg/l.
Controls: Test medium without test substance or other additives (Blank-control).

TEST PROCEDURE AND CONDITIONS
Test type: Static
Test duration: 48 hours
Test vessels: 100 ml, all-glass
Medium: ISO
Number of daphnia: 20 per concentration
Loading: 5 per vessel containing 80 ml medium
Feeding: No feeding
Light: 16 hours photoperiod daily
Aeration: No aeration of the test solutions.
Introduction of daphnids Within 20 minutes after preparation of the test solutions.

MEASUREMENTS AND RECORDINGS
Immobility (including mortality): At 24 hours and 48 hours.
pH and dissolved oxygen: At the beginning and at the end of the test, for the limit concentration and the control.
Temperature of medium: Continuously in a temperature control vessel, beginning at the start of the test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.2 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
Immobility
At the end of the test period undissolved test substance particles were observed in the test concentration, which was due to testing above water solubility. Daphnids however were not disturbed by this precipitation.

Experimental conditions
These test conditions remained within the limits prescribed by the protocol (pH: 6.0-8.5, not varying by more than 1 unit; oxygen: > 7 mg/l at the start, ≥5 mg/l at the end of the test).
The temperature of the test medium was 20.8°C at the start of the test. The temperature continuously measured in a temperature control vessel varied between 20.0 and 21.3°C during the test, and complied with the requirements as laid down in the protocol (18-22°C, constant within 2°C).
Results with reference substance (positive control):
The actual responses in the reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at NOTOX.
The 24h-EC50 was 1.0 mg/l with a 95% confidence interval between 0.94 and 1.2 mg/l.
The 48h-EC50 was 0.74 mg/l with a 95% confidence interval between 0.66 and 0.84 mg/l.
Reported statistics and error estimates:
ACCEPTABILITY OF THE TEST
1. In the control, no daphnia became immobilised or trapped at the surface of the water.
2. The oxygen concentration was ≥ 5 mg/l at the end of the test. Other test conditions (pH and temperature) were maintained within the limits prescribed by the guidelines.
3. The 24h-EC50 (based on the initial concentration) of potassium dichromate was within the range 0.6 mg/l to 1.7 mg/l.

Incidence OF Immobility in the limit test

Loading rate HATCOL 5236 (mg/l)

Vessel number

Number Daphnia exposed

Response at 24h

Response at 48h

Number

Total %

Number

Total %

Blank-control

A

5

0

0

0

0

B

5

0

0

C

5

0

0

D

5

0

0

100

A

5

0

0

0*

0

B

5

0

0*

C

5

0

0*

D

5

0

0*

*Precipitation was observed.

 

pH and oxygen concentrations during the limit test

Loading rate HATCOL 5236 (mg/l)

Start (t=0h)

End (t=48h)

pH

O2

pH

O2

Blank-control

7.9

8.5

8.0

8.7

100

7.8

8.0

7.7

8.1

 

Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate:

Concentration (mg/l)

Number exposed

% immobile

Expected response (%)

24 h

48 h

After 48 hours1

Minimal

Maximal

Blank-control

20

0

0

0

102

0.10

20

0

0

0

10

0.18

20

0

0

0

10

0.32

20

0

0

0

30

0.56

20

0

5

0

100

1.0

20

35

95

40

100

1.8

20

100

100

100

100

1Based on historical date of the previous years (n>60).

2A maximum response of 10% does not invalidate the results of the test.

Validity criteria fulfilled:
yes
Conclusions:
Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h-EC50 for Daphnia magna exceeded the maximum solubility of HATCOL 5236 in water.
Executive summary:

Acute Toxicity Study in Daphnia magna with HATCOL 5236.

The study procedures described in this report were based on the ISO International Standard6341: "Water quality- Determination of the inhibition of the mobility of Daphnia magna Straus-Acute toxicity test, Third edition, 1996-04-01. In addition, the procedures were designed to meet the test methods and validity criteria of the EEC directive 92/69, Part C: Methods for the determination of ecotoxicity, Publication No. L383, December 1992, C.2. "Acute Toxicity for Daphnia", and the DECO guideline No. 202 Part I: "Daphnia sp., Acute Immobilisation Test., Adopted April 4, 1984.

HATCOL 5236 is a clear colourless liquid with a purity of 97.6%. The water solubility of HATCOL 5236 at 20.0 ± 0.8°C was determined to be < 2.0x10e-4 g/l using the flask method.

A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution wasstirred for two days to reach maximum solubility. Afterthe stirring period the mixture was hazy and contained a test substance floating layer. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filer (ca. > 5µm). The filtrate was still slightly hazy.

A limit test was performed exposing twenty daphnids per concentration to a 5 pm filtered solution prepared at a loading rate of 100 mg/l and a blank-control for a maximum of 48 hours.

The test was performed in quadruplicate with 5 daphnids per vessel. Samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples showed that concentrations remained above the solubility limit of HATCOL 5236 (i.e. < 0.2 mg/l) throughout the test.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

HATCOL 5236 did not induce acute immobilisation of Daphnia magna when exposed to a filtered solution prepared at a loading rate of 100 mg/l, corresponding with concentrations abovethe water solubility.

In conclusion: Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h EC50 for Daphnia magna exceeded the maximum solubility of HATCOL 5236 in water.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
17 - 19 Jan 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)- Method: Water Accommodated Fraction (WAF): This WAF was prepared by moderately stirring 100 mg test substance per liter of medium overnight followed by filtration (MILLIPORE AP15 glass fiber filter).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM- Common name: Water flea- Strain: Straus- Length at study initiation (length definition, mean, range and SD): < 2mm- Method of breeding: Parental and young Daphnia were held in 40L glass aquaria. BREEDING- Breeding conditions (same as test or not): Reconstituted water (ISO 6341) was the same as test, prepared with distilled water. The medium in the breeding system was continuously aerated in comparison to the test. - Type and amount of food: 15 g sera micropan (sera GmbH, Heinsberg) homogenized in 1L deionized water- Feeding frequency: Once a day, except weekendsACCLIMATION- Acclimation period: Daphnia, which had been selected in size, were acclimatized to the reconstituted water for 4 hours before introduction into the test media.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Test temperature:
20.0 ± 0.5 °C
pH:
control: 7.3 at 0h, 7.7 at 48h100 mg/l: 7.5 at 0h, 7.7 at 48h
Dissolved oxygen:
control: 6.2 mg/l at 0h, 6.0 mg/l at 48h100 mg/l: 6.0 mg/l at 0h, 5.9 mg/l at 48h
Nominal and measured concentrations:
nominal: 100 mg/L (WAF)
Details on test conditions:
TEST SYSTEM- Test vessel: - Material, size, headspace, fill volume: 100 mL flasks, all-glass, with 50 mL of test medium - Aeration: no- No. of organisms per vessel: 20- No. of vessels per concentration (replicates): 2- No. of vessels per control (replicates): 2TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Reconstituted water (ISO 6341), prepared with distilled water. The ratio of Ca:Mg ions is 4:1 and of Na:K ions is 10:1.- Alkalinity: 0.8 mmol/L- Ca/mg ratio: 4:1- Culture medium different from test medium: No, but test medium was not aerated as the culture medium.- Intervals of water quality measurement: Temperature determined in a control flask at the start and at the end of the test. Oxygen and pH were determined in all vessels prior to the addition of Daphnia to the test medium and at the end of the test.OTHER TEST CONDITIONS- Photoperiod: 16 h a day, supplied by overhead white fluorescent tubes.EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations of immobile Daphnia were made after 24 and 48 h of exposure
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL0
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility

The test substance show no acute toxicity for daphnids under test conditions.

Table 1: Immobilisation of Daphnia magna after 24 and 48 hours

Nominal concentration (mg/l)

Code

Number of individuals per test vessel

Immobile Daphnia (No./vessel)

24h

Immobile Daphnia (No./vessel)

48h

% immobile Daphnia after 24h of exposure

% immobile Daphnia after 48h of exposure

Control
0

A

B

C

20
20
20

0
1
0

0
1
0

0
5
0

0
5
0

100

A

B

20
20

0
1

1
1

0
5

5
5

 

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
03 February to 03 April 2000
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Inconsistent result (test substance film at the highest concentration, number of daphnids trapped at the surface at each concentration)
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water accommodated fractions (WAFs); weighed amounts of test substance expressed as mg/l were mixed with test medium, treated with
ultrasonic waves and stirred for ca. 48 hours in a closed vessel in the dark; the dispersions were allowed to settle overnight until a stable separation
of liquid phases was reached.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
Common name: Water flea
- Strain: Straus
- Age at study initiation (mean and range, SD): < 24 hours

HOUSING
- Housing conditions: Maximum age of the cultures was 4 weeks, renewal of the cultures was after 7 days of cultivation half of the medium twice a
week
- Type and amount of food: A suspension of fresh water algae
- Feeding frequency: Daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20.5-21.8 °C (control)
pH:
7.8-8.0
Dissolved oxygen:
Start (t=0 h): 9.2-9.7
End (t=48 h): 8.8-9.2
Nominal and measured concentrations:
Water Accommodated Fractions (WAFs) prepared at: 10, 18, 32, 56 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass
- Material, size, headspace, fill volume: 80 ml fill volume
- Aeration: none
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO, prepared in milli-RO water
- Culture medium different from test medium: yes, culture medium was medium M7 as prescribed by Elendt-Schneider (Elendt, 1990), trace elements, macro nutrients and vitamins are added to freshly prepared ISO-medium
- Intervals of water quality measurement: pH and dissolved oxygen at the beginning and at the end of the test

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobility at 24 and 48 hours

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: WAFs prepared at 10 and 100 mg/l and dilutions containing 1 and 10% of the 10 mg/l WAF
- Results used to determine the conditions for the definitive study: All daphnids exposed to a loading rate of 100 mg/l (WAF) are immobilised at the end of the test period. No immobility was observed in the lower test concentrations.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
78 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval between 68 and 98 mg/L
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: A number of daphnids trapped at the surface at each concentration. At a test concentration of 18 mg/L daphnids
stucked to the bottom of the test vessel. Before recording of immobility the daphnids were reimmersed in the test solution.
- Mortality of control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At 100 mg/L (WAF) a test substance film was observed after 24 hours of exposure.
- Effect concentrations exceeding solubility of substance in test medium: yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50/LC50: EC50 (48h) of potassium dichromate was 0.45 mg/L (95% fiducial limits of 0.41 - 0.53 mg/L)

At each test concentration daphnids trapped at the surface of the treated test solutions.

At 100 mg/L test concentration the daphnids showed an immobilisation of > 50% (final test: 75% (mean), range finding test: 100%) after 48h exposure. A test substance film was observed after 24 hours of exposure at this highest concentration. The EC50 was stated as 78 mg/l (nominal). Because of the test substance film at the highest concentration and the trapped daphnids at the surface at each concentration, the test is not reliable. The effect at 100 mg/L could result in physical effects caused in undissolved substance.

Table 1: Acute immobilisation of Daphnia magna after 24 and 48 hours

(between brackets: number of daphnids observed trapped at the surface)

Test substance (WAF at x mg/l)

Vessel number

Number Daphnia exposed

Immobilisation at 24h

Immobilisation at 48 h

number

%

number

%

Control

A

B

10

10

0

0

0

0

0

0

0

0

10

A

B

10

10

0 (7)

0 (3)

0

0

1 (1)

0 (2)

10

0

18

A

B

10

10

3 (10)

0 (6)

30

0

0

1 (2)

0

10

32

A

B

10

10

0 (10)

0 (10)

0

0

0

0 (1)

0

0

56

A

B

10

10

0 (6)

0 (5)

0

0

3 (1)

0

30

0

100

A

B

10

10

3 (10)

2 (9)

30

20

7

8

70

80

 

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance Fatty acids, C5-9 tetraesters with pentaerythritol (CAS 67762-53-2). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Qualifier:
according to
Guideline:
other: OECD series on testing and assessment 23, 2000
Principles of method if other than guideline:
Test conducted by preparing WAFs due to low water solubility of the test substance.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: During the final test singular samples for possible analysis were taken from all test concentrations and the control at test start and test end
- Sampling method: 2 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis

At the end of the exposure period, the replicates were pooled at each concentration before sampling. Additionally, reserve samples of 2 mL were taken for possible analysis. If not used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying 2 days of magnetic stirring to ensure maximum dissolution in test medium. The resulting dispersion was left to settle for 1 hour (range-finding test) or 2 hours (final test) were after the Water Accomodated Fraction (WAF) was collected by siphoning for use as highest test concentration. The lower test concentrations were prepared by subsequent dilutions in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The final test solutions were all clear and colourless.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: In-house laboratory culture with a known history. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood
- Age at study initiation: <24 hours, from parental daphnids of more than two weeks old
- Method of breeding: Start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. Maximum age of the cultures is 4 weeks. Renewal of the cultures: after 7 days of cultivation half of the medium twice a week. Temperature of medium (M7, Elendt): 18 - 22 °C. Daphnids werde fed daily with a suspension of fresh water algae.
- Feeding during test: no
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
180 mg/L expressed as CaCO3
Test temperature:
19.8 °C - 20.4 °C
pH:
7.9 - 8.0 (final test)
Dissolved oxygen:
8.7 - 8.9 mg/L (final test)
Nominal and measured concentrations:
combined limit/range finding test: control, 0.1, 1.0, 10 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final test: control, 4.6, 10, 22, 46 and 100% of the WAF prepared at a loading rate of 100 mg/L (nominal)
final test: control, 0.045, 0.18, 0.47, 0.63, and 1.3 mg/L (measured)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, fill volume: glass vessels with a barrier net just below the surface to prevent Daphnids to swim to the surface in order to avoid contact to any undissolved test material, 90-mL, 70 mL fill volume
- Aeration: no
- No. of organisms per vessel: 5 (combined limit/range finding test and final test)
- No. of vessels per concentration (replicates): 4 (final test), 4 replicates at 100% WAF, 2 at 0.1, 1.0, and 10% (combined limit/range finding test)
- No. of vessels per control (replicates): 4 (combined limit/range finding test and final test)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no; M7 medium after Elendt (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33)


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): immobility at 24 hours and 48 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 in combined limit/range finding test, approx. 2 in final test
- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100% of the WAF
- Results used to determine the conditions for the definitive study: The two highest test groups contained a floating layer from 24 hours onwards causing entrapment of a significant number of organisms. Additional microscopic evaluation performed after 24 hours showed that bodies of the daphnids in the highest test group were all covered with test article. Hence, at least part if not all of the effects were of physical nature and caused by exposure to undissolved test material. It was concluded that the test set-up for the final study should include a barrier to prevent organisms getting stuck in the floating layer.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test mat. WAF
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
46 other: % of a WSF prepared at a loading rate of 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test mat. WAF
Basis for effect:
mobility
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: yes

Limit/range finding test: Immobility at 10 and 100% WAF were at respectively 50 and 95% at the end of the test. It should however be recognized that the two highest test groups contained a floating layer from 24 hours onwards causing entrapment of a significant number of organisms. Additional microscopic evaluation performed after 24 hours showed that bodies of the daphnids in the highest test group were all covered with test article. Hence, at least part if not all of the effects were of physical nature and caused by exposure to undissolved test material.

4.8 Samples taken from 1.0 and 100% of the WAF prepared at 100 mg/L were analysed. The initial concentration in the undiluted WAF was 2.2 mg/L. This concentration decreased to 0.22 mg/L after 48 hours. In samples taken from 1.0% WAF, the measured concentration decreased from 0.17 mg/L at the start to a concentration below the limit of detection during the test period. Concentrations in both solutions decreased significantly during the test period, which was expected to be related to the extremely low water solubility (< 6 µg/L).

Analytical results final test: Analysis of the samples taken at the start of the final test showed measured concentrations of 0.27, 0.85, 1.9, 3.6 and 7.0 mg/L in solutions representing 4.6, 10, 22, 46 and 100% of the WSF, respectively. The concentrations decreased during the test period to approximately 3 - 6% of initial. However, measured concentrations in all solutions at the end of the study still exceeded the water solubility that was determined to be less than 6 µg/L. Based on these results, the average exposure concentrations were calculated to correspond with 0.045, 0.18, 0.47, 0.63 and 1.3 mg/L.

Contrary to the combined limit/range-finding test, no daphnids became trapped at the surface despite the fact that initial exposure concentrations in the undiluted WSF were even higher than those measured in the combined limit/range-finding test. This was the result of the use of a physical barrier in combination with placing the light source underneath the vessels. As a consequence, only very few daphnia were immobilised in comparison to the same solutions tested in the combined limit/range-finding test. This confirmed that effects in the range-finding were of physical instead of toxic nature.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50 = 0.3 mg/L (95% CL: 0.28 - 0.35 mg/L

Table: Incidence of immobility in the combined limit/range-finding test

Test group

Fatty acids, C5-9, tetraesters with pentaerythritol

(% WSF*)

Vessel number

 

Number

Daphnia

exposed

Response at 24 h

Response at 48 h

 

number

Total

%

 

number

Total

%

Control

A

B

C
D

5

5

5

5

0

0

0

0

0

0

0

0

0

0

       0.1

A

B

5

5

0

0

0

0

0

0

       1.0

A

B

5

5

0

0

0

0

0

0

     10

A

B

5

 31

0 (4)

0 (2)

0

2 (1)

2

50

   100

A

B

C

D

5

5

5

 61

3 (5)

4 (5)

5 (5)

5 (5)

81

5

4 (1)

5

6

95

* = Test groups represent percentages of a WAF prepared at 100 mg/L

1 = Incorrect number of organisms exposed

( ) = number of Daphnia observed trapped at the surface of test solutions. These organisms were reimmersed into the respective solutions before recording of mobility

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
8 Nov - 30 Nov 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance Decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid (CAS 71010-76-9). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Individual treatments were prepared by adding the appropriate amount of test substance to 2 L of laboratory dilution water in glass aspirator bottles. The test substance was added to the water in the aspirator bottles using stainless steel and glass syringes. The syringes were weighed before and after adding the test substance to determine the actual loading rate. The mixing vessels were closed with foil-covered neoprene stoppers. The mixtures were stirred using a 6.7% (of the static liquid depth) vortex for 23.5 h on magnetic stir plates with Teflon-coated stribars at room temperature. As stirring initiated and after stirring all treatments appeared clear and colourless with the test substance floating at the surface. The mixtures were allowed to settle and equilibrate to test temperature in a waterbath at 17.8 °C for one hour without stirring before removing the aqueous portions (WAFs) through the outlet at the bottom of the vessel for testing.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: in-house culture; original culture was supplied by Aquatic Biosystems, Fort Collins, USA on April 11, 2002
- Age at study initiation: < 24 h
- Feeding during test: no

ACCLIMATION
- Acclimation period: Day 0 cultures were started daily using 8 < 24 h old neonates from culture beakers between 12 and 18 days old, exhibiting <20% adult mortality
- Acclimation conditions. same as test
- Type and amount of food: Pseudokirchneriella subcapitata (~ 4.5E5 cells/mL) and 4 mL of a yeast/salmon starter/ Microfeast mixture per 800 mL
- Feeding frequency: daily (five days per week at a minimum)
- Health during acclimation: The pool of neonates selected for the test had ≤ 10% daily mortality on the experimental start day.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
150 mg/L as CaCO3
Test temperature:
20.1 - 20.9
pH:
7.9 - 8.1
Dissolved oxygen:
7.3 - 8.3 mg/L
Nominal and measured concentrations:
Nominal: 62, 132, 251, 503 and 1072 mg/L loading rate WAF
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type: closed with ground glass stoppers
- Material: glass, size: 125 mL, fill volume: ~140 mL (no headspace)
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was prepared from UV-sterilized, deionized well water that was treated and distributed throughout the testing facility via PVC and stainless steel pipes.
- Ca/mg ratio: 1.2:1
- Alkalinity: 96 mg/L as CaCO3
- Conductivity: 500 µmhos
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water quality measurements (pH, dissolved oxygen, temperature) were performed on a sub-sample of the WAFs from each treatment and control on day 0 and on a composite of the replicates in each treatment at termination.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light/ 8 h dark
- Light intensity: Daylight intensity ranged from ~184 - 233 lux during full daylight periods of the study.

EFFECT PARAMETERS MEASURED: Immobilisation was recorded after 24 and 48 h test duration. In addition, observations for normal or abnormal behaviour/ appearance were made.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: The toxicity of the "water-accomnodated fraction" of the test substance was tested at different loading levels. The WAFs were prepared by adding the appropriate amount of test substance to dilution water in glass aspirator bottles. The vessels were closed and mixed on magnetic stir plates with Teflon coated stirbars for 23 h and 50 minutes. The treatments were allowed to settle for 1 h and five minutes after mixing. Two replicates at each loading level were prepared containing 5 organisms. This study was not conducted according to GLP.
- Test concentrations: 10, 100 and 1000 mg/L loading rate WAFs
- Results used to determine the conditions for the definitive study: No mortality at any loading level was recorded during a exposure time of 48 hours.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 1 072 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: none

TOC analysis confirmed the low water solubility of the test substance in water. The total carbon content of the treatment is only slightly higher than that of the control. Thus, the results of the TOC analysis can not be used for the determination of an effect concentration. Thus, the effect concentration is related to the nominal concentration.

Table 1: TOC analysis of the test substance in the test medium

TOC [mg]

Loading rate

0 h

48 h

Control

0.4127

0.4102

62

0.4716

0.4246

132

0.4349

0.4330

251

0.3856

0.3966

503

0.4420

0.4097

1072

0.4415

0.4079

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21 Jul - 24 Aug 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance Mixed esters of pentaerythritol, technical grade, with isooctanoic and C8/C10 fatty acids (CAS 189200-42-8). According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control, 1000 mg/L
- Sampling method: Samples were removed from the test solutions on day 0 and at termination.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Individual treatment solutions (WAF) were prepared by adding the appropriate amount of the test substance to 1 L of dilution water in 1 L size glass aspirator bottles. The vessels were closed with cotton-gauze stoppers to minimize evaporation and/or volatilazition. The solutions were mixed at <10% vortex on magnetic stirplates with Teflon® coated stirbars for approximately 24 hours. All treatment levels appeared clear with test substance floating on the surface. After mixing, the solutions were allowed to settle for approximately one hour before the WAFs were removed through the outlet at the bottom of the aspirator bottle.
- Evidence of undissolved material: No test substance insolubility was observed in any of the test chambers.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Source: in-house culture
- Age at study initiation: < 24 hold, taken from 16 d old parents
- Method of breeding: Daphnids are kept in 1 L glass culture vessels with dilution water (BW3 at 20 ± 2 °C) supplemented with vitamin B12 and selenium.
- Feeding during test: none

ACCLIMATION
- Acclimation period: not specified
- Acclimation conditions: same as test
- Type and amount of food: Selenastrum capricornutum and a 6 µL/mL yeast/ salmon starter/ cereal leaves mixture
- Feeding frequency: daily
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
190 mg/L as CaCO3
Test temperature:
21.2 - 22.0 °C
pH:
7.6 - 8.0
Dissolved oxygen:
7.0 - 8.3 mg/L
Nominal and measured concentrations:
Nominal: 62.5, 125, 250, 500 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask sealed with ground glass stoppers
- Material: glass; Size: 125 mL; Fill volume: 140 mL of solution
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory dilution water, a mixture of carbon filtered well water and water dialyzed by reverse osmosis supplemented with 1 µg/L vitamin B12 and 2 µg/L selenium, aged and aerated ≥ 24 h prior to introduction of the test substance
- Total organic carbon: 1.528 ppm
- Alkalinity: 57 mg/L as CaCO3
- Conductivity: 370 µmhos
- Culture medium different from test medium: no
- Intervals of water quality measurement: Water quality measurements (pH, dissolved oxygen and temperature) were performed on a subsample of each treatment on Day 0 and a composite of the replicates at termination.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark with a gradual intensity conversion between periods
- Light intensity: 640.56 - 642.82 lux during full daylight periods

EFFECT PARAMETERS MEASURED: Immobilisation was recorded after 24 and 48 h test duration.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAF)
Basis for effect:
mobility
Details on results:
- Mortality of control: No mortality observed.
- Effect concentrations exceeding solubility of substance in test medium:

The quality of the dilution water used in culture and testing is monitored at weekly, monthly and semi-annual intervals. There are no known contaminants in the feed or water believed to be at levels high enough to interfere with this study based on contaminant analysis and/or historical data.

The TOC analysis indicates that the level of test substance in the WAF was at or near the level of organic carbon in the control. This result confirms the low solubility of the test substance in water and the unsuitability to use the measured concentrations for the determination of the effect value. Thus, the effect value is related to the nominal concentration.

Table 1: Measuremnt of the test concentration

Loading level [mg/L]

Total organic carbon [mg/L]*

Day 0

 “new”

Day 2

“old”

Control

19 ± 0.3

10 ± 0.8

1000

6.6 ± 0.3

16 ± 1.0

*: triplicate TOC measurements on each sample

Table 2: Immobilisation of Daphnia

Loading level [mg/L]

Percent immobilisation

24 h

48 h

Control

0

0

62.5

0

5

125

0

0

250

0

0

500

0

0

1000

0

5

Description of key information

Key value determined in static acute toxicity study in accordance with OECD guideline 202 and EU test standard C2.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.1 mg/L

Additional information

HATCOL 3331

Acute Toxicity Study in Daphnia magna with HATCOL 3331.

The batch of HATCOL 3331 tested was a clear colourless liquid with a purity of 97.3% and the substance was not completely soluble in test medium at the concentration tested. The water solubility of Hatcol 3331 at 20.3 ± 0.8°C was determined to be < 2.0x10E-4 g/l, according to the flask method (NOTOX Project 365052). The partition coefficient (n-octanol/water), Pow, was determined to be ≥ 5.1*10E6 (log Pow ≥ 6.7) at 20.3 ± 0.8°C (NOTOX Project 365085).

All solutions at loading rates of 1.0 mg/l and higher were prepared separately. These supersaturated solutions were magnetically stirred for two days to ensure maximum solubility in test medium. The resulting solution was colourless, but contained a floating layer and test substance particles. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filter (ca. > 5 µm). The filtrate was clear and colourless.

The project was started with a limit test, exposing daphnia to a filtrate (ca. > 5 µm) prepared at a loading rate of 100 mg/l and a blank-control. The test was performed in duplicate with 5 daphnids per vessel and samples for analysis were taken at the start and the end of the test. The analytical results showed that the average exposure concentration was higher than the solubility limit of HATCOL 3331 (i.e. < 0.2 mg/l). At the end of the test, 45% of the daphnids exposed to the filtrate were immobilized. Since concentrations were all below 1.0 mg/l, a final test was performed using a range of concentrations nominally spaced by a factor of 10.

A final test was performed exposing daphnia to filtrates (ca. > 5 µm) prepared at loading rates of 1.0, 10 and 100 mg/l and a 10-fold dilution of the filtrate at 1.0 mg/l. Samples for analysis taken at the start and the end of the test showed that the average exposure concentration in the 1.0 mg/l filtrate approximated the water solubility (i.e. < 0.2 mg/l), while average exposure concentrations in the 10 and 100 mg/l filtrates were maintained above the water solubility of HATCOL 3331.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

HATCOL 3331 did not induce acute immobilisation of Daphnia magna at the concentration obtained in a filtered solution prepared at a loading rate of 100 mg/l, corresponding to an average exposure concentration above the water solubility, i.e. 1.1 -1.4 mg/l after 48 hours of exposure (NOEC). Also, no immobilisation was observed at concentrations approximating the water solubility.

In conclusion: Due to the very low solubility of HATCOL 3331 in water, concentration levels that might be toxic for daphnia could not be reached. Therefore, the 48h-EC50 exceeded the maximum solubility of HATCOL 3331 in test medium.

 

HATCOL 5236

Acute Toxicity Study in Daphnia magna with HATCOL 5236.

HATCOL 5236 is a clear colourless liquid with a purity of 97.6%. The water solubility of HATCOL 5236 at 20.0 ± 0.8°C was determined to be < 2.0x10e-4 g/l using the flask method.

A stock solution was prepared at a loading rate of 100 mg/l. This supersaturated solution was stirred for two days to reach maximum solubility. After the stirring period the mixture was hazy and contained a test substance floating layer. Collection of the water phase by siphoning or centrifugation was not an option considering the specific gravity of the test substance. After the stirring period the mixture was therefore filtered through a paper filer (ca. > 5µm). The filtrate was still slightly hazy.

A limit test was performed exposing twenty daphnids per concentration to a 5 pm filtered solution prepared at a loading rate of 100 mg/l and a blank-control for a maximum of 48 hours.

The test was performed in quadruplicate with 5 daphnids per vessel. Samples for analytical confirmation of actual exposure concentrations were taken at the start and at the end of the test.

Analysis of the samples showed that concentrations remained above the solubility limit of HATCOL 5236 (i.e. < 0.2 mg/l) throughout the test.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

HATCOL 5236 did not induce acute immobilisation of Daphnia magna when exposed to a filtered solution prepared at a loading rate of 100 mg/l, corresponding with concentrations above the water solubility.

In conclusion: Owing to the extremely low solubility of HATCOL 5236 in water, concentration levels toxic for crustaceans could not be reached. Therefore, the 48h EC50 for Daphnia magna exceeded the maximum solubility of HATCOL 5236 in water.

CAS 11138 -60 -6

The study was conducted with Daphnia magna and according to OECD 202. The results provided an EL 50 (48h) > 100 mg/L (WAF loading rate).

CAS 71010-76-9

The short-term toxicity of decanoic acid, mixed esters with heptanoic acid, octanoic acid, pentaerythritol and valeric acid to aquatic invertebrates was investigated according to OECD 202 using Daphnia magna as test organism under static condition (Blattenberger, 2006). The test solutions were prepared by adding the appropriate amount of test substance with subsequent stirring and sampling of the aqueous portions (WAFs) through the outlet at the bottom of the vessels. Loading rate WAFs of 62, 132, 251, 503 and 1072 mg/L were tested. No immobilisation was observed in any treatment and the control throughout the test period of 48 h. Hence, the 48 h-EL50 is determined to be > 1072 mg/L based on the nominal test concentration.

 

CAS 67762-35-2

The short-term toxicity of fatty acids, C5-9 tetraesters with pentaerythritol to aquatic invertebrates was investigated in a study according to OECD 202 using Daphnia magna as test organism under static conditions (Migchielsen, 2012). The WAFs were prepared by adding the appropriate amount of test substance with subsequent stirring and sampling of the aqueous portions A nominal test concentration of 100 mg/L, corresponding to a measured concentration of 1.3 mg/L, was tested. No immobilisation was observed in the treatment throughout the test period of 48 h. Hence, the 48 h-EL50 is determined to be > 100 mg/L based on the nominal test concentration and > 1.3 mg/L based on the measured concentration. It can therefore be concluded that the test substance will not exhibit short-term effects to aquatic invertebrates up to the limit of water solubility.

 

CAS 11138-60-6

Short term toxicity of fatty acids, C8-10 (even numbered), di-and triesters with propylidynetrimethanol, was investigated according to OECD 202. All measured results from 48 -hour toxicity studies indicate the test substance is of low toxicity to Daphnia magna. The study conducted with CAS 11138-60-6 (Häner, 2007) determined EL 50 (48h) > 100 mg/L (WAF loading rate).

 

CAS 189200-42-8

Short short term toxicity of fatty acids, C8-10 mixed esters with dipentaerythritol, isooctanoic acid, pentaerythritol and tripentaerythritol was investigated according to OECD 202. All measured results from 48 -hour toxicity studies indicate the test substance is of low toxicity to Daphnia magna. The study (Febbo (1995) observed an EL50 > 1000 mg/L, WAF loading rate.

 

CAS 68424-30-6

Short-term study with the target substance tetraesters from esterification of pentaerythritol with pentanoic, heptanoic and isononanoic acids rated as RL 4 due to insufficient documentation. In this study using Daphnia magna as test organism under static conditions (Windeatt, 1995) a 48 h-EC50 > 10 mg/L based on the nominal test concentration was determined. It can therefore be concluded that the test substance will not exhibit short-term effects to aquatic invertebrates up to the limit of water solubility.