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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2015 to 12 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guideline No. 202. All validity criteria were fulfilled
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
5 March 2015
Specific details on test material used for the study:
PHYSICO-CHEMICAL PROPERTIES
- Specific Gravity at 20 °C: 0.8-0.9
Analytical monitoring:
yes
Details on sampling:
- Chemical analyses: Single sample for analysis were taken from the control and all test concentrations at the start of the test (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for the fresh media at t=0 h and t=24 h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each test vessel and 5.5 L of test water was added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vortex in the centre extending maximally around 10 % vessel depth from the top to the bottom of the vessel. After 24 ± 2 h of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 h before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test tubes that were immediately sealed with screwcaps after introduction of daphnids. No small bubble was observed in the test tubes. The test solution was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions (WAFs).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C/Daphnia/day. The water was changed at least once per week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Total water hardness was approximately 250 mg/L (as CaCO3)
Test temperature:
20.9-21.9 °C (average value: 21.4 °C)
pH:
8.09-8.38
Dissolved oxygen:
8.01-8.50 mg/L
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal concentrations: 1.0, 1.3, 1.7, 2.3 and 3.0 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item).
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessel each completely filled with test solution and without headspace as soon as possible after addition of the WAFs into test tubes.
- Chemical analyses were taken from additional replicates with daphnids in order to avoid disturbing the daphnids at t=24h and to prevent potential test item losses on opening test tubes: two additional series for sampling for analysis of test concentrations from the freshly prepared solutions (t=0 h and t=24 h fresh); and another for sampling from the 24 h old solutions (t=24 h old). The sampling for analysis of the control and test concentrations at t=48 h was performed directly from one of the 4 remaining replicates with daphnids after reading of the immobilisation test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (Elendt M4 medium), as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 h. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h) from all test concentrations and control.
- Temperature of Medium: Measured continuously in a vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.32
- Range finding study: Daphnids were exposed to the nominal test concentrations of 1.0, 2.0, 5.0 and 10 mg/L and to a control.
- Results used to determine the conditions for the definitive study: The per cent immobility was 0, 0, 30, 100 and 100 (24 h) and 0, 0, 100, 100 and 100 (48 h) at 0, 1.0, 2.0, 5.0 and 10 mg/L, respectively. Based on the results of a range-finding test, the following nominal concentrations (spaced by a factor of approximately 1.32): 1.0, 1.3, 1.7, 2.3 and 3.0 mg/L were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 1.7 - < 2.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2.821 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 2.622 - 3.127 mg test item/L
Details on results:
- After 24 h of exposure, immobilisations rates were 0 % at 1.0, 1.3 and 1.7 mg/L (loading), 10 % at 2.3 mg/L (loading) and 65 % at 3.0 mg/L (loading).
- After 48 h of exposure, immobilisations rates were 0 % at 1.0 mg/L (loading), 5 % at 1.3 mg/L (loading), 15 % at 1.7 mg/L (loading) and 100 % at 2.3 and 3.0 mg/L (loading).
- The 24 h EC50 including the 95 % confidence interval using Probit-analysis was determined by the computer program ToxRat. At t=48 h, an EC50 value was also determined by the software but due to lack of concentration/response data this value (1.834 mg/L) could not be valid. However, given immobilisations rates at the end of the test, the 48 h EC50 value should be estimated higher than 1.7 mg/L (loading) and lower than 2.3 mg/L (loading).
Results with reference substance (positive control):
On September 10, 2015 (most recent test), the 24 h-EC50 was 0.94 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24 h-EC50: 0.6 to 2.1 mg/L) at this time.
Reported statistics and error estimates:
The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Analytical results

Although every effort was made to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), some constituents were lost over the study period. However, analytical monitoring of dl-limonene (sum of 2 isomers, representing 47 % of the mixture) revealed losses not exceeding 38 % of the initial concentration and not exceeding 20 % of the initial concentration at one relevant loading rate (2.3mg/L). Moreover, the measured concentrations of beta-phellandrene (29.6 % of the mixture) were overall stable and within ± 20 % of the initial concentration (except at 3.0 mg/L with losses ca. 25 % the first day and ca. 40 % the second day). Thus, since the major constituents of the mixture were generally maintained within or close to ± 20 % of the nominal concentration throughout the test and since the study was carried out using WAFs, it was considered acceptable to express the biological results based on nominal loading values.

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 hours in the final test

 

Nominal concentration* (mg/L)

Replicate

Number of daphnids exposed

Response at 24 h

Response at 48 h

Number

Total %

Number

Total %

Control

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

1.0

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

1.3

1

5

0

0

0

5

2

5

0

1

3

5

0

0

4

5

0

0

1.7

1

5

0

0

1

15

2

5

0

1

3

5

0

1

4

5

0

0

2.3

1

5

0

10

5

100

2

5

1

5

3

5

0

5

4

5

1

5

3.0

1

5

2

65

5

100

2

5

5

5

3

5

4

5

4

5

2

5

  * WAF prepared at the given loading rate

Water quality parameter values throughout the test:

Test conditions remained within the limits prescribed by the study plan (pH: 6.0-9.0, not varying by more than 1.5 units;oxygen:≥ 60 % of the air-saturation valueat the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.9 and 21.9 °C throughout the test (average value: 21.4 °C), and complied with the requirements as laid down in the study plan (20 ± 2 °C, constant within 1 °C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was≥ 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:
yes
Conclusions:
The 48h-EC50 of the test item was estimated between 1.7 and 2.3 mg/L (loading). The NOEC was determined to be 1.3 mg/L (loading).
Therefore, based on log Kow > 4, Reaction mass of beta-phellandrene and d-limonene and l-limonene is classified in chronic category 2 according to CLP regulation (EC) No. 1272/2008.
Executive summary:

A study was performed to assess the acute toxicity to Daphnia magna of test item REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE according to OECD Guideline 202 with GLP compliance under semi-static conditions. 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 1.0, 1.3, 1.7, 2.3 and 3.0 mg/L and to a control. The immobility of the daphnids was determined in a semi-static 48h test by visual observation after 24 and 48h. The concentrations of the test item, represented by analytical monitoring of the main constituents d- and l-limonene, and beta-phellandrene, were determined by chemical analyses at the start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h).

 

Although every effort was made to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), some constituents were lost over the study period. However, analytical monitoring of dl-limonene (sum of 2 isomers, representing 47% of the mixture) revealed losses not exceeding 38% of the initial concentration and not exceeding 20% of the initial concentration at one relevant loading rate (2.3 mg/L). Moreover, the measured concentrations of beta-phellandrene (29.6 % of the mixture) were overall stable and within ± 20% of the initial concentration (except at 3.0 mg/L with losses ca. 25% the first day and ca. 40% the second day). Thus, since the major constituents of the mixture were generally maintained within or close to ± 20% of the nominal concentration throughout the test and since the study was carried out using WAFs, it was considered acceptable to express the biological results based on nominal loading values.

After 24h of exposure, immobilisations rates were 0% at 1.0, 1.3 and 1.7 mg/L (loading), 10% at 2.3 mg/L (loading) and 65% at 3.0 mg/L (loading). After 48h of exposure, immobilisations rates were 0% at 1.0 mg/L (loading), 5% at 1.3 mg/L (loading), 15% at 1.7 mg/L (loading) and 100% at 2.3 and 3.0 mg/L (loading).

24h EC50: 2.821 mg/L (95% Cl 2.622-3.127 mg/L)

48h EC50:> 1.7-< 2.3mg/L

Therefore, based on nominal concentrations, the 48h-EC50 value was estimated between 1.7 and 2.3 mg/L (loading). The NOEC was determined to be 1.3 mg/L (loading).

Therefore, based on log Kow > 4, Reaction mass of beta-phellandrene and d-limonene and l-limonene is classified in chronic category 2 according to CLP regulation (EC) No. 1272/2008.

Description of key information

Based on nominal concentrations, the 48-h EL50 value of Reaction mass of beta-phellandrene and d-limonene and l-limonene was estimated between 1.7 and 2.3 mg/L (loading).

Therefore, based on log Kow > 4, Reaction mass of beta-phellandrene and d-limonene and l-limonene is classified in chronic category 2 according to CLP regumation (EC) No 1272/2008.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
1.7 mg/L

Additional information

A reliable study was available to assess the acute toxicity to Daphnia magna of test item Reaction mass of beta-phellandrene and d-limonene and l-limonene according to OECD Guideline 202 with GLP compliance under semi-static conditions. 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 1.0, 1.3, 1.7, 2.3 and 3.0 mg/L and to a control.

The major constituents of the mixture were generally maintained within or close to ± 20% of the nominal concentration throughout the test and since the study was carried out using WAFs, it was considered acceptable to express the biological results based on nominal loading values.

After 24 h of exposure, immobilisations rates were 0% at 1.0, 1.3 and 1.7 mg/L (loading), 10% at 2.3 mg/L (loading) and 65% at 3.0 mg/L (loading). After 48 h of exposure, immobilisations rates were 0% at 1.0 mg/L (loading), 5% at 1.3 mg/L (loading), 15% at 1.7 mg/L (loading) and 100% at 2.3 and 3.0 mg/L (loading).

Therefore, based on nominal concentrations, the 48-h EC50 value was estimated between 1.7 and 2.3 mg/L (loading). The NOEC was determined to be 1.3 mg/L (loading).

Therefore, based on log Kow > 4, Reaction mass of beta-phellandrene and d-limonene and l-limonene is classified in chronic category 2 according to CLP regulation (EC) N° 1272/2008.