Tall oil, potassium salt

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2012 to 18 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each loading rate WAF test group (replicates R1 - R4 pooled) at 0 (fresh media), 24 (old and fresh media) and 48 hours (old media) for quantitative analysis. Samples were stored at approximately -20 °C prior to analysis.
Duplicate samples were taken and stored at approximately -20 °C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
In view of the difficulties associated with the evaluation of aquatic toxicity of complex mixtures that are poorly soluble in water and the permitted auxiliary solvents and surfactants, a modification of the standard method for the preparation of aqueous media was performed, exposing the organisms to a Water Accommodated Fraction (WAF) of the test material. Using this approach, aqueous media were prepared by mixing the test material with water for a prolonged period. Previous experience has shown that a preparation period of 24 hours is sufficient to ensure equilibration between the test material and water phase. At the completion of mixing and following a 1 hour settlement period, the test material phase was separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test material and/or leachates from the test material). Exposures are typically expressed in terms of the original concentration of test material in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test material in the WAF; however in this study, at the request of the Sponsor, results have been expressed in terms of mean measured test material concentrations and nominal loading rates.

20, 36, 64, 112 and 40 mg of test material were each separately weighed onto a glass slide and suspended in the water column of 20, 20, 20, 20 and 4 litres of reconstituted water to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates respectively. After the addition of the test material, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of dispersed test material was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75 to 100 mL discarded) to give the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that the glass wool plug had removed all of the dispersed test material.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and flake food suspension.
Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Approximate theoretical total hardness of 250 mg/L as CaCO₃.

Test temperature:

21 °C

pH:

7.6 to 8.1

Dissolved oxygen:

8.2 to 9.5 mg O₂/L, expressed as 92 to 107 % Air Saturation Value (ASV).

Nominal and measured concentrations:

Nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass jars.
- Type: Closed; vessels were covered to reduce evaporation.
- Fill volume: 200 mL of test preparation.
- Aeration: Test vessels were not aerated. Dilution water was aerated prior to study initiation, until the dissolved oxygen concentration was approximately the air saturation value.
- No. of organisms per vessel: 5 daphnids.
- No. of vessels per concentration: Four replicates.
- No. of vessels per control: Four replicates.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was prepared by adding 25 mL aliquots of each of the following solutions to each litre (final volume) of deionised water.
CaCl₂.2H₂O (11.76 g/L); MgSO₄.7H₂O (4.93 g/L); NaHCO₃ (2.59 g/L) and KCl (0.23 g/L).
- Conductivity: < 5 µS cm^-1
- pH: 7.8 ± 0.2, adjusted as necessary with NaOH or HCl.
- Intervals of water quality measurement: Water temperature, dissolved oxygen and pH were recorded in the fresh media at 0 and 24 hours and the old media at 24 and 48 hours.

OTHER TEST CONDITIONS
- Renewal of media: The test preparations were renewed at 24 hours during the exposure period.
- Photoperiod: A 16 hour light/ 8 hour darkness cycle was maintained, with 20 minute dawn and dusk transition periods.
- Light intensity: Ranged from 492 to 654 lux.

EFFECT PARAMETERS MEASURED: Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours. Immobile daphnids were defined as those which were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS
- Nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.
In view of the difficulties associated with the evaluation of aquatic toxicity of complex mixtures that are poorly water soluble, a modification of the standard method for the preparation of aqueous media was performed, exposing the organisms to a Water Accommodated Fraction (WAF).

Reference substance (positive control):

yes

Remarks:

potassium dichromate (0.32, 0.56, 1.0, 1.8 and 3.2 mg/L)

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

2.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % confidence limits of 0.57 to 7.2 mg/L loading rate WAF.

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loading rate WAF

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Loading rate WAF

Details on results:

Cumulative immobilisation data are given in Table 1.
Microscopic observations carried out on immobilised daphnids showed that no test material was adhered to appendages.
Analysis of the immobilisation data by the probit method (Finney, 1971) at 24 and 48 hours based on the nominal loading rates gave the following results:
Time EL50 95 % Confidence limits
(h) (mg/L loading rate WAF) (mg/L loading rate WAF)
24 6.2 -
48 2.4 0.57 - 7.2
The Lowest Effect Loading rates after 24 and 48 hours were considered to be 3.2 and 1.8 mg/L loading rate WAF respectively. The No Observed Effect Loading rates after 24 and 48 hours exposure were 1.8 and 1.0 mg/L loading rate WAF respectively.
The slopes and their standard errors of the response curves at 24 and 48 hours were 1.9 (SE = 1.2) and 4.5 (SE = 1.3) respectively.

OBSERVATIONS ON TEST MATERIAL SOLUBILITY
Observations on the test media were carried out during the mixing and testing of the WAFs.
At the start of the mixing period the 1.0, 1.8, 3.2, 5.6 and 10 mg/L loading rates were observed to be clear, colourless water columns with test material visible on the glass slide suspended in the water column. After 23 hours stirring and a 1 hour standing period the 1.0 and 1.8 mg/L loading rates were observed to be clear, colourless water columns with particles of test material dispersed throughout, while the 3.2, 5.6 and 10 mg/L loading rates were observed to be cloudy dispersions with particles of test material dispersed throughout. Visual examination of the WAFs showed that test material was dispersed throughout the water columns and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed test material. During the test the 1.0, 1.8, 3.2 and 5.6 mg/L loading rates were observed to be clear, colourless solutions, whilst the 10 mg/L loading rate was observed to be a slightly cloudy solution.

CHEMICAL ANALYSIS OF TEST LOADING RATES
Chemical analysis of the freshly prepared test media at 0 hours showed measured test concentrations to range from 83 to 101 % of nominal. A slight decline in measured concentration was observed at 24 hours in the range of 67 to 86 % of nominal. Analysis of the freshly prepared test media at 24 hours showed measured concentrations to range from 65 to 83% of nominal. A decline in measured concentration was observed in the old test media in the range of 48 to 95 % of nominal.
During the preparation of the WAFs, dispersed test material was observed in the water columns and filtration through glass wool was required in order to remove this; however, measured concentrations of near nominal were obtained. This was due to the presence of multiple components in the test material, with one that was soluble being analysed.

Duplicate samples were analysed from the 10 mg/L loading rate WAF which highlighted an erroneous result, in this case the duplicate result was used in the calculation of the time weighted mean measured concentrations. However, for the 1.0 mg/L loading rate at 24 and 48 hours, the duplicate analysis result seems to be erroneous. The duplicates were analysed at this concentration as lower than expected levels were observed originally; however, the original result shows a decline over time which is consistent with the other concentrations. It was therefore considered appropriate to use the original result to calculate the time weighted mean measured concentrations.

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the results were within the normal range.
- 24 hour EC50: 0.75 mg/L (0.56 to 1.0 mg/L 95 % confidence limits)
- 48 hour EC50: 0.45 mg/L (0.42 to 0.48 mg/L 95 % confidence limits)
- 24 hour NOEC: 0.56 mg/L
- 48 hour NOEC: 0.32 mg/L

Table 1 Cumulative Immobilisation Data

Nominal Loading Rate (mg/L)	Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
	24 Hours						48 Hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0	0	0
1.8	0	0	0	0	0	0	5	0	1	0	6	30
3.2	5	5	2	2	14	70	5	5	5	3	18	90
5.6	3	4	2	0	9	45	5	5	4	3	17	85
10	3	4	1	2	10	50	5	5	5	5	20	100

R1 to R4 = Replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, exposure of Daphnia magna to the test material gave a 48 hour EL50 value based on nominal loading rates of 2.4 mg/L; 95 % confidence limits 0.57 - 7.2 mg/L loading rate WAF.

Executive summary:

The acute toxicity of the test substance to Daphnia magna was investigated in accordance with OECD Guideline 202 and EU Method C.2 under GLP conditions. Due to the low aqueous solubility and complex nature of the test substance, it was prepared as a water accommodated fraction (WAF). Following preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to WAFs of the test substance over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours at a temperature of approximately 21°C under semi-static test conditions. The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours. Chemical analysis of the freshly prepared test media at 0 hours showed measured test concentrations to range from 83 to 101 % of nominal. A slight decline in measured concentration was observed at 24 hours in the range of 67 to 86 % of nominal. Analysis of the freshly prepared test media at 24 hours showed measured concentrations to range from 65 to 83% of the nominal concentrations. Under the conditions of this study, the 48 h EL50 was determined to be 2.4 mg/L WAF (Harris, 2013).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.4 mg/L

Additional information

The acute toxicity of the test substance toDaphnia magnawas investigated in accordance with OECD Guideline 202 and EU Method C.2 under GLP conditions. Due to the low aqueous solubility and complex nature of the test substance, it was prepared as a water accommodated fraction (WAF). Following preliminary range-finding tests, twenty daphnids (4 replicates of 5 animals) were exposed to WAFs of the test substance over a range of nominal loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L for 48 hours at a temperature of approximately 21°C under semi-static test conditions. The number of immobilisedDaphniaand any adverse reactions to exposure were recorded after 24 and 48 hours. Chemical analysis of the freshly prepared test media at 0 hours showed measured test concentrations to range from 83 to 101 % of nominal. A slight decline in measured concentration was observed at 24 hours in the range of 67 to 86 % of nominal. Analysis of the freshly prepared test media at 24 hours showed measured concentrations to range from 65 to 83% of the nominal concentrations. Under the conditions of this study, the 48 h EL50 was determined to be 2.4 mg/L WAF (Harris, 2013).