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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-07-18 to 2010-08-10
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 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Loading rates: 0 (Control), 2.5, 10 and 50 mg/L loading rate. The vessels used for Exposure Concentration Analysis, including the Control and WAFs, were kept under the same conditions as the test vessels but without Fish present.

- Sampling method: Samples of the control and WAFs were taken for TOC analysis (TOC = Total Organic Carbon) and GC-MS analysis (GC-MS = Gas Chromatography - Mass Spectrometry) from separate vessels without Fish at the start and end of test periods 0 - 24 h and 72 - 96 h to assess the stability of exposure concentrations.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method:The test item is a complex mixture with a low water solubility. To avoid adverse physical effects of undissolved or emulsified test material on the Fish, the Fish Test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in tap water.

In a preliminary study with a test item of comparably low water solubility (Sylvablend 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 Fish Test were prepared by stirring various amounts of the test item in tap water for 24 h with magnetic stirrers. The WAFs were prepared in 20 L solid-glass fish tanks. The test item was weighed onto watch glasses that afterwards were placed into the solid-glass fish tanks. The fish tanks were then filled with tap water and closed immediately with a glass plate. 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 WAFs were then siphoned with a glass tube and transferred into the test vessels, i.e. 20 L solid-glass fish tanks, that were closed with a glass plate immediately after the transfer.

For the Fish Test Water Accommodated Fractions were prepared in 20 L solid-glass fish tanks and replenished after each successive 24-h exposure period (i.e. semi-static test) for a total exposure period of 96 hours. Preparation of WAFs started the previous day and the WAFs were used for the Fish Test the next day. The WAFs obtained using this method were clear.

The Control media (tap water) for the Fish Test were treated in the same way as the WAFs.

- Controls: Dilution water

- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test substance was not fully soluble at the tested loading rate. However the method of preparing the WAFs was designed to mimimise exposure to undissolved test material.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM

- Common name: Zebrafish

- Source: - Source: Max-Planck-Institut für Entwicklungsbiologie, Tübingen

- Length at study initiation: 2.0 +/- 1 cm

- Feeding during test: none

ACCLIMATION

- Acclimation period: >12 days

- Acclimation conditions: same as test

- Type and amount of food: not reported

- Feeding frequency: not reported

- Health during acclimation (any mortality observed): mortality < 1 %
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
8.97 ºdH
Test temperature:
23 +/- 2.0ºC
pH:
8.2-8.3
Dissolved oxygen:
89-100% ASV
Salinity:
not applicable
Nominal and measured concentrations:
Nominal loading rates: 0(Control), 2.5, 5.0, 10, 25 and 50 mg/L WAF

TOC-values 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 2.5 mg/L, 10 mg/L and 50 mg/L lie within or very close to the confidence-limits (± 0.3 mg C/L) of these measurements. Therefore, the WAFs were considered to remain approximately stable over the 96-h test period.

Analysis of the Control medium and the WAFs in the Fish Test revealed, that the total concentrations of fingerprint constituents, determined by GC-MS analysis, decreased to 82 % of the initial value at test loading rate 2.5 mg/L at observation period 0 - 24 h and to 90 % of the initial value at observation period 72 - 96 h. Total concentrations of fingerprint components decreased to 64 % of the initial value at test loading rate 10 mg/L at observation period 0 - 24 h and were stable with 103 % of the initial value at observation period 72 - 96 h. Total concentrations of fingerprint components decreased to 88 % of the initial value at test loading rate 50 mg/L at observation period 0 - 24 h.

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 test results are reported with reference to nominal loading rate.
Details on test conditions:
TEST SYSTEM

- Test vessel:

- Type: glass aquaria, closed with glass plates

- Material, size, headspace, fill volume: Glass, 20 litre, 18 litre fill volume

- Aeration: none

- Renewal rate of test solution: daily

- No. of organisms per vessel: 7

- No. of vessels per concentration (replicates): 1

- No. of vessels per control (replicates): 1

- Biomass loading rate: < 1.0 g Fish/L


TEST MEDIUM / WATER PARAMETERS

-Origin: Wasserversorgung Reutlingen (Bodenseewasser)

- Total hardness: 8.97 °dH

- Carbonate hardness: 6.98°dH,

- Nitrate: 4.4 mg/L, Nitrite: < 0.005 mg/L,

- Ammonium: < 0.01 mg/L

- Hg/Cd/Pb/Cu/Ni: < 0.001 mg/L

- Conductivity: 304 µS/cm

- Intervals of water quality measurement: daily


OTHER TEST CONDITIONS

- Adjustment of pH: no

- Photoperiod: Gro-Lux fluorescent tubes (14 h light, 10 h dark)


EFFECT PARAMETERS MEASURED: daily observations of mortality and behaviour


TEST CONCENTRATIONS

- Results of range-finding test: none

- Spacing factor for definitive test loading rates: 2-2.5
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water-accommodated fraction loading rate
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
water-accommodated fraction loading rate
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: none

- Mortality of control: 0

- Other adverse effects control: none
Reported statistics and error estimates:
Determination of the LL50 (LL = Lethal Loading) of the lethality in the Fish Test was accomplished using Probit-analysis (Finney-method, lognormal distribution; confidence limit 95 %, significance level: 0.05). The NOELR was determined directly from the raw data.

Table 1. Test results - percentage mortality for each observation period

Time [h]

Control

2.5 mg/L

5.0 mg/L

10 mg/L

25 mg/L

50 mg/L

 

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

Number

[%]

24

0/7

0

0/7

0

0/7

0

0/7

0

0/7

0

1/7

14.3

48

0/7

0

0/7

0

0/7

0

0/7

0

0/7

0

7/7

100

72

0/7

0

0/7

0

0/7

0

0/7

0

0/7

0

7/7

100

96

0/7

0

0/7

0

0/7

0

0/7

0

1/7

14.3

7/7

100

Table 2. Results of TOC analysis of test media (mg/L)

Period

Control

2.5 mg/L

10 mg/L

50 mg/L

0-24 h start

1.3

1.0

*

2.9

0-24 h end

0.8

0.7

1.2

3.0

72-96 h start

1.5

1.6

1.9

-

72-96 h end

1.4

1.3

1.8

-

Table 4. 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 - 24 start

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

Control

0 - 24

end

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

Control

72 -96 start

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

Control

72 - 96 end

0.214**

< 0.367*

0.227**

< 0.151*

0.212**

< 1.17

2.5 mg/L

0 - 24 start

2.27

< 0.367*

0.227**

0.625

0.463

< 3.95

2.5 mg/L

0 - 24

end

1.74

0.636

0.227**

0.437

0.212**

3.25

2.5 mg/L

72 -96 start

1.51

1.03

0.227**

0.596

0.414

3.78

2.5 mg/L

72 - 96 end

1.47

0.754

0.227**

0.564

0.372

3.39

10 mg/L

0 - 24 start

14.0

34.0

2.78

9.49

4.11

64.4

10 mg/L

0 - 24

end

7.72

23.4

1.86

5.57

2.36

40.9

10 mg/L

72 -96 start

6.11

34.5

3.82

11.7

5.07

61.2

10 mg/L

72 - 96 end

6.18

36.5

3.32

12.1

5.01

63.1

50 mg/L

0 - 24 start

43.1

405

31.6

122

47.9

649

50 mg/L

0 - 24

end

42.6

396

20.2

80.3

33.6

573

 

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 96-hour LL50 value of 29 mg/L and a NOELR of 5 mg/L have been determined for the effects of the test substance on mortality of Danio rerio. The fish were exposed to water-accommodated fractions of the substance.

Description of key information

A 96h-LL50 of 29 mg/l and NOELR of 5 mg/l have been determined for the effect of the test substance on mortality of Danio rerio. The substance had a sulfur content of 0.02%. A 96h- LL50 of 45.1 mg/l and NOELR of 5 mg/l have been determined for the same endpoint in a second test conducted on a sample of the substance with a sulfur content of 3.6%. It is therefore concluded that the two samples were equally toxic to fish.

The above data are consistent with the data available for UVCBs hydrocarbons, terpene-processing by-products (CAS 68956-56-9) which share a large number of their constituents with TOPP. The 96h-LL50 of around 5 mg/L was reported for this substance.

The 96h-LC50 of alpha-pinene, a constituent of block 1 of TOPP, has been determined as 0.30 mg/l.

Key value for chemical safety assessment

LC50 for freshwater fish:
29 mg/L

Additional information