Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1999-07-27 to 1999-11-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline compliant GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
as at 1984
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
as at 1992
Qualifier:
according to
Guideline:
ISO 6341 15 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Version / remarks:
as at 1996-04-01
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dl-Lactone
- Physical state: white crystalline mass
- Analytical purity: 100.0 %
- Lot/batch No.: 805046
- Expiration date of the lot/batch: 01 May 2002
- Stability under test conditions: Stable under storage conditions
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 and 100 mg/L dose groups were tested, samples from other doses kept deep frozen for confirmatory analysis if needed
- Sampling method: 10 mL from the approximate center of the vessel
- Sample storage conditions before analysis: tested on sampling day

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: semi static, as test item is hydrolytical unstable in water (see study Brekelmans (1999) / Rep. No. 257568 / key (Hydrolysis as a function of pH) in chapter 5.1.2); renewal of test solutions after 24 hours of exposure
- Controls: medium control, historical positive control (regular reference test with K2Cr2O7)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): all solutions were clear

MEDIA
- all chemicals in analytical grade
- water: freshly prepared ultra-pure water (tap water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water; Millipore Corp., Bedford, Mass., USA):
- masses in mg/L
- ISO-MEDIUM:
CaCl2.2H2O 293.8
MgSO4.7H2O 123.3
NaHCO3 64.8
KCI 5.8
- MEDIUM M7:
trace elements, macro nutrients and vitamins added to freshly prepared ISO-medium to reach the following concentrations:
Trace elements (mg/L):
B 0.125
Fe 0.05
Mn 0.025
Li, Rb and Sr 0.0125
Mo 0.0063
Br 0.0025
Cu 0.0016
Zn 0.0063
Co and J 0.0025
Se 0.0010
V 0.0003
Na2EDTA.2H20 2.5
Macro nutrients (mg/L):
Na2SiO3.9H2O 10.0
NaNO3 0.27
KH2PO4 0.14
K2HPO4 0.18
Vitamins (µg/L):
Thiamine 75.0
B12 1.0
Biotin 0.75

Hardness: 250 mg/I expressed as CaCO3
pH: 8.0 ± 0.2 after aeration.

Preparation of test solutions
- Start with stock solutions at 100 mg/L
- careful mixing sufficient to dissolve the test substance in the test medium
- lower test concentrations for the range-finding test prepared by subsequent dilutions of one of the stocks in test medium

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Age at study initiation (mean and range, SD): < 24 h
- Method of breeding:
Start of 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, constant within ± 1°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).
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: yes, breeding in same medium and same laboratory conditions
- Acclimation conditions (same as test or not): yes

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
no

Test conditions

Hardness:
250 mg/I expressed as CaCO3
Test temperature:
20.3 - 20.7 °C
pH:
7.8 - 8.0 (see table 2)
Dissolved oxygen:
7.8 - 9.2 (see table 2)
Salinity:
not applicable
Nominal and measured concentrations:
nominal: 100 mg/L
actual: ca. 130 mg/L (see table 3)
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass beaker
- Type: open
- Material, size, fill volume: all-glass, 100 mL size, 80 mL fill volume
- Aeration: no active aeration
- Renewal rate of test solution (frequency/flow rate): once after 24 h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2


TEST MEDIUM / WATER PARAMETERS
- see above under "details on test solutions (MEDIA)
- Culture medium different from test medium: no
- Intervals of water quality measurement: at the beginning, after 24 h of exposure and at the end of exposure

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilisation: animals not able to swim within 15 seconds after gentle agitation of the test vessel

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
pretest: 10
main test: limit test with 100.0 mg/L
- Justification for using less concentrations than requested by guideline: limit test at highest requested concentration
- Range finding study
Test concentrations: 0.1, 1.0, 10.0, 100.0 mg/L
Results used to determine the conditions for the definitive study: no immobilisation at 100 mg/L
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 (regular reference test, batch no. 148 K15548364, obtained from Merck, Darmstadt, Germany.

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24
Dose descriptor:
EC50
Effect conc.:
> 130 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 130 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: no
- Observations on body length and weight: not applicable
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- see table 4 for details
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50/LC50: 24 h-EC50 = 1.2 mg/L, 48 h-EC50 = 0.62 mg/L (0.52 - 76 mg/L 95 % CL); see table 5 for details

Any other information on results incl. tables

- Table 1: Incidence of immobility in the range-finding test

Concentration
DL-LACTONE
(mg/I)

Number
Daphnia
exposed

Response at 24 h

Response at 48 h

number

%/

Number

%

0.1

10

0

0

0

0

1.0

10

0

0

0

0

10

10

0

0

0

0

100

10

0

0

0

0

- Table 2: pH and oxygen concentrations during the limit test (main test)

Concentration (mg/L)

Start (t=0 h)

t=24h old

t=24h fresh

End (t=48 h)

DL-LACTONE

pH

oxygen

pH

oxygen

pH

oxygen

pH

oxygen

Nominal / Actual

 

 

 

 

 

 

 

 

Blank-control

7.9

9.2

8.0

7.8

8.0

9.1

8.0

7.9

100 / 130

7.8

9.2

7.9

7.8

7.9

9.1

7.8

7.8

- Table 3: Concentrations of DL-LACTONE in test medium (final test).

Time of
sampling
[hours]

Date of
sampling
[dd-mm-yy]

Date of
analysis
[dd-mm-yy]

Concentration

Nominal
[mg/L]

Analysed (1)
[mg/L]

Relative to
nominal [%]

0

24-08-99

24-08-99

0

n.d.

n.a.

0

 

 

100

144

144

0

 

 

100 (2)

140

140

24 old

25-08-99

25-08-99

0

n.d.

n.a.

 

 

 

100

123

123

24 fresh

25-08-99

25-08-99

0

n.d.

n.a.

 

 

 

100

138

138

(1) Mean of duplicate analysis. The maximum deviation between the responses was calculated for each sample and was < 10%.

(2) Reserve sample.

n.d.: not detected.

n.a.: not applicable.

Analysis of the samples taken during the limit test showed that the measured concentration was 144 mg/L in the freshly prepared 100 mg/L test solution. An extra sample was analysed at t=0 to check whether the high recovery was correct. The measured concentration in the reserve sample was 140 mg/L.

The measured concentration after 24 hours was 123 mg/L.

Hence, the measured concentration remained within a 20% window during the 24-hour period between refreshment.

Preparation of test solutions proved to be repeatable as the concentration measured in the freshly prepared 100 mg/L solution after 24 hours of exposure was 138 mg/I.

In conclusion: the average exposure concentration during the study was approximately 130 mg/L. No clear explanation can possibly be given for the relative high recoveries measured.

- Table 4: Acute immobilisation of daphnia after 24 and 48 hours in the limit test (main test)

Concentration (mg/L) DL-LACTONE

Vessel Number

Number
Daphnia

Response at 24 h

Response at 48 h

 

 

 

 

Nominal / Actual

 

exposed

number

%

number

°A

Blank-control

A

10

0

0

0

0

 

B

10

0

0

0

0

100 / 130

A

10

0

0

0

0

 

B

10

0

0

0

0

ACCEPTABILITY OF THE TEST

1. In the controls, no daphnia became immobilised or trapped at the surface of the water.

2. The analytical program provided clear evidence that the actual concentrations during the 24-hour period between refreshment had been maintained at more than 80 % of the initial concentration.

3. Further, all test conditions remained within the ranges prescribed by the protocol.

- Table 5: Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate

Concentration (mg/L)

Number Exposed

% immobile

24h

48h

Expected response (%) After 48 hours (1)

 

Minimal

Maximal

0.00

10

0

0

0

10 (2)

0.10

10

0

0

0

10

0.18

10

0

0

0

10

0.32

10

0

0

0

30

0.56

10

0

30

0

100

1.0

10

10

100

40

100

1.8

10

100

100

100

100

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

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

The actual responses in this 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 in the laboratory.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present test RS Pantolactone (named DL-LACTONE in the report) induced no acute immobilisation of Daphnia magna at or below 100 mg/L, corresponding with an average measured concentration of 130 mg/L (NOEC). Hence, the 48h-EC50 was above 130 mg/L, the
maximum concentration tested.
Executive summary:

RS-Pantolactone (named DL-Lactone in the report) was tested for its short-term toxicity to aquatic invertebrates with Dapnia magna according to OECD TG 202. The study procedures described in the report fulfil also the ISO International Standard 6341: "Water quality - Determination of the inhibition of the mobility of Daphnia magna Straus - Acute toxicity test, Third edition, 1996-04-01.

RS-Pantolactone was completely soluble in test medium at the concentrations tested. The complete project was performed under semi-static test conditions with renewal of test solutions after 24 hours of exposure as the test item proved to be unstable in water at pH values of 7 and 9 in the hydrolysis test performed at NOTOX (Project 257568, IUCLID5 chapter 5.1.2).

A range-finding test was followed by a limit test exposing Daphnia for a maximum of 48 hours to a concentration of 100 mg/I and a blank-control. The test was performed in duplicate with 10 daphnia per vessel. Samples for analysis of actual exposure concentrations were taken from the freshly prepared solutions at the start and the 24-hour old and freshly prepared solutions after 24 hours of exposure.

Analysis of the samples taken during the limit test showed that the measured concentration remained within a 20% window during the 24-hour period between refreshment. The average measured concentration corresponded with approximately 130 mg/I.

RS-Pantolactone induced no acute immobilisation of Daphnia magna at or below 100 mg/L, corresponding with an average measured concentration of 130 mg/L (NOEC).

Hence, the 48h-EC 50 was above 130 mg/L, the maximum concentration tested.

By expert judgement it is concluded that L-Pantolactone will get the same results:

L-Pantolactone: 48h EC 50 above 130 mg/L.