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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:
2010-06-29 to 2010-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without restrictions. The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Loading rates: 0 (Control), 1.0, 5.0 and 25 mg/L

- Sampling method: Samples of the control and WAFs of the Definite Test were taken for TOC analysis (TOC = Total Organic Carbon) and GC-MS analysis (GC-MS = Gas Chromatography - Mass Spectrometry) from separate vessels without Daphnia at 0 h and 48 h.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: The test item Wood Turpentine is a complex mixture with a low water solubility. To avoid adverse physical effects of undissolved or emulsified test material on the animals, the Range-Finding and Definite Test with Wood Turpentine were carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in Daphnia-medium.

In a preliminary study with a test item of comparably low water solubility (Sylva-blend TM PF 40, STZ No. 01-05-001; study sponsor Arizona Chemical), the durations of the stirring and phase separation phases of WAF preparation were investigated. Water Accommodated Fractions were prepared and analysed following a range of mixing and settling periods to ensure optimal equilibrium and phase separation of the test item in the WAFs. Quantitation of dissolved constituent concentrations was achieved by TOC analysis (TOC = Total Organic Carbon).

Taking into account the results of this preliminary WAF-study, the WAFs for the Range-Finding and Definite Test with Wood Turpentine were prepared by stirring various amounts of the test item in Daphnia-medium for 24 h with magnetic stirrers. The WAFs were prepared in closed glass vessels. The test loading rates of Wood Turpentine were weighed on weighing scoops, that afterwards were placed into the glass vessels. The vessels were then filled with Daphnia-medium and 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. To ensure this, the vortex developed at the surface by stirring was set at approximately 10 % of the water depth. After stirring for 24 h the WAFs were allowed to stand for 1 h before use to facilitate phase separation. The extracts gained with this method were clear.

The control media (Daphnia-medium) for the Range-Finding and Definite Test were treated in the same way as the WAFs.

The vessels used for Exposure Concentration Analysis, including the Control and WAFs of the Definite Test, were kept under the same conditions as the test vessels.

- Controls: Daphnia culture medium
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Strain: Clone 5

- Source: Daphnia magna (Clone 5) originally obtained from the German Environment Ministry

- Age at study initiation (mean and range, SD): <24 hours

- Method of breeding: Laboratory stock culture

- Feeding during test: none


ACCLIMATION

- Acclimation conditions: same as test

- Type of food: algae (Desmodesmus subspicatus)

- Photoperiod: 14 h light/10 h dark (Fluorescent tubes)

CULTURE MEDIUM

Stock solutions in deionised water:
CaCl2 x 2 H2O: 11.76 g/L
KCl: 0.23 g/L
MgSO4 x 7 H2O: 4.93 g/L
NaHCO3: 2.59 g/L

25 ml aliquots of each stock solution were mixed and then topped up to 1 L with deionised water. The water was aerated until the concentration of oxygen complied with the saturation value of atmospheric oxygen. The total of Ca- and Mg-ions was 2.5 mmol/L, the ratio of Ca/Mg was 4:1, the ratio of Na/K was 10:1. The total alkalinity of the solution was 0.8 mmol/L, the pH-value was 7.8 +/- 0.2.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
2.5 mmol/L
Test temperature:
20+/-2.0ºC
pH:
7.8-8.0
Dissolved oxygen:
7.9-8.1 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
Loading rates: 0 (Control), 0.5, 1.0, 2.5, 5.0, 10 and 25 mg/L

TOC-values of the Definite Daphnia Test with 6 test loading rates confirmed the low water solubility of the test item (Table 2). The deviations of the TOC-values in the control and at test loading rates of 1.0 mg/L, 5.0 mg/L and 25 mg/L lie within the confidence-limits ( ± 0.3 mg C/L) of these measurements. Therefore the WAFs were considered to remain stable over the 48-h test period.

Analysis of the Control medium and the WAFs in the Definite Daphnia Test revealed, that the total concentrations of fingerprint components, determined by GC-MS analysis, decreased to 69.7 % of initial values at test loading rate 1.0 mg/L, to 78.3 % of initial values at test loading rate 5.0 mg/L and
to 76.7 % of initial values at test loading rate 25 mg/L over 48 h (Table 3).

Quantitative GC-MS-values were definitely lower compared to TOC-values. This is caused by the fact that only fingerprints of the sample were detected by GC-MS analysis whilst TOC analysis detected Total Organic Carbon as a sum parameter.

The results are interpreted and expressed relative to nominal loading rates.
Details on test conditions:
TEST SYSTEM

- Test vessel:

- Type (delete if not applicable): flasks with ground-glass stoppers

- Material, size, fill volume: glass, 50 ml with 50 ml of test medium

- Aeration: none

- Renewal rate of test solution (frequency/flow rate): static test

- No. of organisms per vessel: 5

- No. of vessels per concentration (replicates): 4

- No. of vessels per control (replicates): 4

- Biomass loading rate: 1 daphnia/10 ml

TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: demineralized water with added salts, macro-nutrients and trace elements. Comparable to that described in OECD Guideline 202.

- Alkalinity: 0.8 mmol/l

- Ca/mg ratio: 4:1

- Culture medium different from test medium: no

- Intervals of water quality measurement: Start of test and then after 24 and 48 hours

OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: 16 h light, 8 h dark

- Light intensity: not reported

EFFECT PARAMETERS MEASURED: Observations of immobilised daphnids, daphnids trapped at the surface, weak daphnids and carapace moults were made at least three times per day throughout the test.

TEST CONCENTRATIONS

- Spacing factor for definitive test loading rates: 2.0 and 2.5

- Range finding test: The definitive test loading rate was set on the basis of a range-finding test conducted at loading rates of 1, 10 and 100 mg/L which showed that that there were no effects on mobility at loading rates of 1 and 10 mg/L but 100% immobilisation at a loading rate of 100 mg/L.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
ca. 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water-accommodated fraction loading rate
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
6.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water-accommodated fraction loading rate
Basis for effect:
mobility
Details on results:
- Immobilisation of control: 0

- Other adverse effects control: none

- Abnormal responses: none

- Effect concentrations exceeding solubility of substance in test medium: The tested loading rates exceeded the aqueous solubility of the substance.
Reported statistics and error estimates:
The EL50 (EL = Effective Loading) value was calculated using Probit-analysis (Finney-method, lognormal distribution; confidence limit 95 %, significance level: 0.05). The NOELR was determined by comparison of the responses at each loading rate with the control validity criterion for immobilisation (≤ 10 % immobilisation).

Table 1. Test results

 

Control

0.5 mg/L

1.0 mg/L

2.5 mg/L

5.0 mg/L

10 mg/L

25 mg/L

 

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Immobilisation at 24 h

0/20

0

0/20

0

0/20

0

0/20

0

0/20

0

12/20

60

20/20

100

Immobilisation at 48 h

0/20

0

0/20

0

1/20

5

2/20

10

1/20

5

18/20

90

20/20

100

Moults at 48 h

18/20

90

15/20

75

15/20

75

14/20

70

15/20

75

6/20

30

3/20

15

Living animals at the surface at 48 h

 

0/20

 

0

 

0/20

 

0

 

0/20

 

0

 

0/20

 

0

 

0/20

 

0

 

0/20

 

0

 

0/20

 

0

 

 

Table 2. Results of TOC analysis of test media

Time [h]

Control

1.0 mg/L

5.0 mg/L

25 mg/L

0

0.1

0.1

0.6

3.0

48

0.3

0.2

0.5

2.7

 

 

Table 3. Results of GC-MS analysis of test media

Vessel

Time [h]

α-Terpineol
[µg/L]

α-Pinene
[µg/L]

β-Pinene
[µg/L]

3-Carene
[µg/L]

Limonene
[µg/L]

Total

[µg/L]

Control

0

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

Control

48

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

1.0 mg/L

0

6.13

15.3

13.7

1.87

5.26

42.2

1.0 mg/L

48

4.22

12.0

8.79

1.22

3.14

29.4

5.0 mg/L

0

32.4

80.2

102

8.80

34.5

258

5.0 mg/L

48

27.5

72.1

73.3

5.68

23.0

202

25 mg/L

0

147

856

748

88.6

242

2081

25 mg/L

48

95.6

854

311

89.3

245

1596

 

Remarks:

*   Limit of detection

**  derived from equation of the calibration line (axis intercept); no signal was measured in the controls for α-Terpineol, β-Pinene and Limonene

     Quantitative GC-MS-values are based on results and/or limits of detection

 

Validity criteria fulfilled:
yes
Conclusions:
A 48 h EL50 value of 6.4 mg/L and NOELR of circa. 1 mg/L have been determined for the effects of the test substance on mobility of Daphnia magna.

Description of key information

A 48h-EL50 value of 6.4 mg/l and NOELR of circa. 1 mg/l have been determined for the effects of a sample of TOPP on mobility of Daphnia magna. In tests with two samples of TOPP, containing 0.02% and 3.6% of sulfur, corresponding EL50 and NOELR values of 8.8 and 2.5 mg/l and 6.8 and 2.5 mg/l were determined for the same endpoint. It is therefore concluded that all three samples were equally toxic to D. magna.

Data are also available for alpha- and beta-pinene, which are constituents of block 1 of TOPP. The 48h-EC50 was determined as 0.48 and 1.25 mg/l respectively.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
6.4 mg/L

Additional information