Tris(2-hydroxyethyl)ammonium salts of tall-oil fatty acids

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

REG (EC) No. 440/2008, May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 100, 50, 25, 12.5, 6.25 mg/L as WAF and control
- Sampling intervals: start and end of the first and the last renewal period
- Sampling method: samples were taken from test vessels containing fish
- Sample storage conditions before analysis: samples taken at the start of the first and last renewal period were stored for one day after processing (freeze-drying)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAFs)
Supersaturated solutions were prepared by dispersing/dissolving the test item into the test medium (filtered tap water) at nominal concentrations of 100, 50, 25, 12.5 and 6.25 mg/L. These dispersions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at the test temperature. The resulting dispersions were opalescent. The non-dissolved test material was removed by filtration through a 0.22-µm filter to provide the WAFs. The filtrates were clear and transparent.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: “Black Molly” Fish Farm, Veszprém, Hungary (for the pre-tests) and Ágnes Németh (for definitive test), Veszprém, Hungary
- Length at study initiation (mean and range, SD): 3.0 ± 0.14 cm (2.7-3.3 cm, n= 42)
- Weight at study initiation (mean and range, SD): 0.38 ± 0.03 g (0.34-0.41 g, n= 42)
- Feeding during test: none

ACCLIMATION
- Acclimation period: > 12 days
- Acclimation conditions (same as test or not): same water quality (circulated and filtered tap water)
- Type and amount of food: commercial fish food (TetraMin staple food for ornamental fish, Tetra GmbH, Germany) at 2-10 mL per feeding.
- Feeding frequency: three times per week, until 24 hours before the start of the test
- Health during acclimation (any mortality observed): The fish were healthy. There was no mortality of the population for seven days before the test

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

203 mg/L as CaCO3 (measured)

Test temperature:

21.1-22.0 °C

pH:

7.08-7.52

Dissolved oxygen:

82-98% air saturation value

Nominal and measured concentrations:

nominal loading rates: 100, 50, 25, 12.5, and 6.25 mg/L as WAF
first renewal period (0-24 h):
- freshly prepared test media: 3.99, 3.13, 2.38, 1.57, and 0.11 mg/L
- aged test media (24 h): 1.36, 1.41, 0.47, 0.25 mg/L, and < LOQ (0.08 mg/L).

second renewal period (72-96 h; the two highest loading rates (100 and 50 mg/L) were not analysed since all fish were dead at that time):
- freshly prepared test media: 2.10, 1.13, and 0.30 mg/L
- aged test media (24 h): 0.18, 0.14 mg/L, and < LOQ (0.08 mg/L).

geometric mean measured concentrations: 2.33, 2.10, 0.81, 0.50, mg/L and 0.09 mg/L.

biological results are based on the nominal loading rates.

Details on test conditions:

TEST SYSTEM
- Test vessel: aquaria, containing 5 litres of medium
- Type: not reported (the test material is not volatile)
- Aeration: not reported (oxygen concentration was sufficient during exposure)
- 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: 0.48 - 0.57 g body weight/L (mean 0.53 ± 0.03 g bw/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: circulated and filtered tap water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and sub-lethal effects; recorded at approx. 3, 6, 24, 48, 72 and 96 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: nominal 100, 10, 1, and 0.1 mg/L
- Results used to determine the conditions for the definitive study: 100% and 25% mortality after 96 hours at 100 and 10 mg/L respectively; no mortality at the lower test concentrations

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence limits: 19-41 mg/L; results are expressed in terms of loading rate

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: results are expressed in terms of loading rate

Duration:

96 h

Dose descriptor:

LOELR

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: one dead fish after 96 h; results are expressed in terms of loading rate

Duration:

96 h

Dose descriptor:

LL0

Effect conc.:

6.25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: results are expressed in terms of loading rate

Duration:

96 h

Dose descriptor:

LL100

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: results are expressed in terms of loading rate

Details on results:

- Behavioural abnormalities: at the 72-hour observation, one fish showed swimming problems at the nominal loading rate of 25 mg/L; no abnormalities observed at any other concentration and/or time
- Mortality of control: zero
- Other adverse effects control: none

For cumulative mortality data Table 1.

Reported statistics and error estimates:

The 24, 48, 72 and 96 hours LL50 values were calculated by Probit analysis with 95% confidence limits using SPSS PC+ software.
The NOELR, LOELR, LL0 and LL100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Table 1: Cumulative mortality data:

 

Nominal loading rate (mg/L)	Cumulative mortality [number dead of fish] *
	3 h	6 h	24 h	48 h	72 h	96 h
Control	0	0	0	0	0	0
6.25	0	0	0	0	0	0
12.5	0	0	0	0	0	1
25	0	0	0	0	0**	1
50	0	0	0	6	7	-
100	0	0	1	7	-	-

*:  initial population = 7 fish

**: one fish with swimming problems

Validity criteria fulfilled:

yes

Remarks:

no mortality of control animals; concentration of dissolved oxygen in all test vessels >60% saturation

Conclusions:

The test material is toxic to freshwater fish with a 96-hour LL50 between 10 and 100 mg/L (calculated value: 28 mg/L).

Description of key information

96-h LC50 (based on nominal loading rates) 28 mg/L for Brachydanio rerio (new name Danio rerio) (OECD 203, EU C.1)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

28 mg/L