Octadecanoic acid, reaction products with diethylenetriamine and urea, acetates

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

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: FAT #: 93580/A
Batch: 0022357000
Purity: 94.3 %
Expiry date :15 December 2015
Description at Room Temperature: White-yellowish waxy solid
Storage Conditions: Room temperature, in the dark

Analytical monitoring:

yes

Details on sampling:

- Concentrations: one limit test concentration based on a loading rate of 100 mg/L
- Sampling method: For the characterization of the water accommodated fraction (WAF) in this semi-static test, duplicate samples were taken from each treatment at the start of each renewal periods (Days 0, 1, 2 and 3). At the end of these renewal periods (Days 1, 2, 3 and 4), additional samples were taken for determination of the stability of the test item during the renewal periods of 24 hours. All samples were taken from the approximate center of the aquaria without mixing of the test media.
- Sample storage conditions before analysis: All samples a were analyzed immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For preparation of the WAF with the loading rate of 100 mg/L, 600 mg of the test item (dosed amounts: 600.3 to 600.5 mg) was mixed into 6 litres of test water. First, the dispersion was treated ultrasonically for 15 minutes. Then, the dispersion was stirred for 96 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. The long stirring time was chosen in order to dissolve a maximum amount of the different components of the sparingly soluble test item and its degradation products in the dispersion. After stirring, the dispersion was left to settle for about 60 minutes to separate undissolved test material from the test water (as far as possible). Thereafter, the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC20, pore size 0.20 µm) after preconditioning the filter material with about 200 mL of test medium to avoid test item loss in the filter. For the filtration at the first three preparations days (0, 1 and 2) the Schleicher & Schuell membrane filter, Type NC20, pore size 0.20 μm with a diameter of 100 mm was used whereas at the last preparation day (3) the same filter type was used but with a diameter of 50 mm. The undiluted filtrate was tested as a WAF. The undiluted filtrate was tested as a WAF.
The stirring period of 96 hours was chosen according to the results of pre-experiments which showed that the solution equilibrium was reached after this time. In these pre-experiments the same test item concentrations were analytically measured in the dispersion after stirring for 3, 24 and 96 hours. The test medium was freshly prepared just before introduction of the fish (= start of the exposure and prior to each test medium renewal).
- Eluate: test water
- Differential loading: not applicable since only one loading rate was used
- Controls: test water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Source: breeding culture at Harlan Laboratories
- Age at study initiation (mean and range, SD): not reported
- Length at study initiation: 2.7 ± 0.11 cm (Mean ± SD)
- Weight at study initiation: 0.15 ± 0.02 g (Mean ± SD)
- Method of breeding: Normal fish breeding conditions according to the needs of the fish species
- Feeding during test: no

ACCLIMATION
- Acclimation period: 14 days without medication, 7 days under test conditions of water and temperature
- Acclimation conditions: same as in test
- Type and amount of food: during holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).
- Feeding frequency: appropriate amounts and appropriate intervals according to the density of fish in the aquaria
- Health during acclimation (any mortality observed): During holding and acclimatization, no fish died in the test fish batch and all fish were healthy.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Test terminated after 96 hrs

Hardness:

125 mg/L as CaCO3

Test temperature:

21 °C throughout the study

pH:

7.1 in all new and aged media

Dissolved oxygen:

7.8-8.6 in new media at 0 hours
8.2-8.6 in old media at 24 hours
7.6-8.5 in new media at 24 hours
8.4 in old media at 48 hours
8.2-8.6 in new media at 48 hours
8.5-8.6 in old media at 72 hours
8.0-8.6 in new media at 72 hours
8.5 in old media at 96 hours

Salinity:

not applicable

Nominal and measured concentrations:

100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass test vessels
- Type: open
- Material, size, headspace, fill volume: glass, 8.5 L, 4.8 L
- Aeration: slight aeration
- Renewal rate of test solution (frequency/flow rate): semi-static test with daily renewal of test medium
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 7 fish per 4.8 L = 1.05 g per 4.8 L = 0.219 g per L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Total organic carbon: not reported
- Particulate matter: not reported
- Metals:
CaCl2 × 2H2O: 1.0 mmol/L = 147 mg/L
MgSO4 × 7H2O: 0.25 mmol/L = 61.5 mg/L
NaHCO3: 0.38 mmol/L = 32.5 mg/L
KCl: 0.038 mmol/L = 2.9 mg/L
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: 0.4 mmol/L
- Ca/mg ratio: 1:4
- Conductivity: not reported but calculable from the known salt concentrations
- Culture medium different from test medium: not during the acclimation phase
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: a 16 hour light to 8-hour dark photoperiod, with a 30-minute transition period
- Light intensity: 140 to 480 Lux

EFFECT PARAMETERS MEASURED: The test fish were observed for mortality and visible abnormalities after approximately 3, 24, 48, 72 and 96 hours test duration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: no since limit test
- Range finding study: no
- Results used to determine the conditions for the definitive study: water solubility limit of the test item (approximately 1.4 µg/L, determined during pre-experiments within this study)

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 38 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: The results were based on the loading rate of 100 mg/L and on the mean measured concentration of 38 μg/L (calculated as arithmetic mean of the geometric means of the test item conc. measured at the start and the end of each test medium renewal period).

Duration:

96 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 38 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: loading reate of 100 mg/L resulted to geometric mean measured concentration of 36 µg/L
- Effect concentrations exceeding solubility of substance in test medium: geometric mean measured concentration of 38 µg/L above the water solubility of the test item

Results with reference substance (positive control):

Not measured

Reported statistics and error estimates:

The NOEC/NOEL and LC0/LL0 were determined directly from the raw data. The LOEC/LOEL, LC100/LL100 and LC50/LL50 at the observation times could not be quantified due to the absence of a toxic effect of the test item at the tested loading rate.
For each test medium renewal period, the mean concentration was calculated as geometric mean of the test item concentrations measured at the start and the end of the test medium renewal period [Sachs, 1984].

Sublethal observations / clinical signs:

Table 1 Analytical results

Sampling Day / Sample Age	Loading Rate Test Item	Measured Concentration of Test Item			Sample Preparation Factor	Determined Concentration of Test Item
		Sample 1	Sample 2	Average
[day/h]	[mg/L]	[µg/L]	[µg/L]	[µg/L]		[µg/L]
0/0	Control	*	*	*	0.06	<LOQ
	100	17.2	17.0	17.1	0.18	3.08
1/24	Control	*	*	*	0.06	<LOQ
	100	13.8	10.3	12.1	0.18	2.17
1/0	Control	*	*	*	0.06	<LOQ
	100	13.6	10.5	12.0	0.18	2.16
2/24	Control	*	*	*	0.06	<LOQ
	100	20	20.9	20.4	0.06	1.23
2/0	Control	*	*	*	0.06	<LOQ
	100	10.6	-	n.a.	0.18	2.68
	100	-	25.5	n.a.	0.06
3/24	Control	*	*	*	0.06	<LOQ
	100	14.5	15.4	14.9	0.06	0.895
3/0	Control	*	*	*	0.06	<LOQ
	100	11.6	11.3	11.4	12.6	144
4/24	Control	*	*	*	0.06	<LOQ
	100	10.5	13.6	12.1	12.6	152

*: the measured concentration was below the lowest calibration concentration

LOQ: 0.118 µg test item/L

n.a.: not applicable

Table 2 Biological results

Treatment / Loading rate [mg/L]	Mean measured concentration [µg/L]	Number of abnormal and dead fish / number of dead fish
		Type of visible abnormalities
		Observation time
		3 hours	24 hours	48 hours	72 hours	96 hours
Control	--	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
100	36	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
LC50* [µg/L]		>38	>38	>38	>38	>38

*: based on mean measured concentration

Validity criteria fulfilled:

yes

Conclusions:

FAT #: 93580/A had no acute toxic effects on zebra fish up to its solubility limit in test water under the present test conditions.

Executive summary:

The acute toxicity of FAT #: 93580/A to zebra fish (Danio rerio) was determined in a 96 hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992).

A limit test was performed in compliance with the test guidelines to demonstrate that water accommodated fractions (WAFs) of the test item have no toxic effect on Danio rerio up to the loading rate of 100 mg/L. Thus, the only concentration tested was a WAF with the loading rate of 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

For preparation of the test medium, a dispersion of the test item with the loading rate of 100 mg/L was prepared. The dispersion was ultrasonic treated for 15 minutes and continuously stirred at room temperature for 96 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersion was filtered through a membrane filter (0.20 μm pore size) either with a diameter of 100 mm (days 0, 1 and 2) or with a diameter of 50 mm (day 3). The undiluted filtrate was tested as a WAF.

The analytically measured test item concentrations (based on the sum of six main components) in the test medium with the loading rate of 100 mg/L were 3.08 µg/L (day 0), 2.16 µg/L (day 1), 2.68 µg/L (day 2) and 144 µg/L (day 3) at the start of the test medium renewal periods. At the end of the test medium renewal periods the measured test item concentrations were 2.17 µg/L (day 1), 1.23 µg/L (day 2), 0.895 µg/L (day 3) and 152 µg/L (day 4). The very high measured concentrations on Day 3 and 4 lead to a mean measured concentration (38 μg/L) far above the determined water solubility limit of the test item in the test water (approximately 1.4 μg/L, determined during pre-experiments within this study). Therefore the results represent a worst case scenario. One possible reason for the observed differences in measured test item concentrations could be the usage of a filter with a smaller diameter (50 mm instead of 100 mm) during the last exposure interval.

The reported biological results were based on the loading rate of 100 mg/L and on the mean measured concentration of 38 µg/L (calculated as arithmetic mean of the geometric means of the test item concentrations measured at the start and the end of each test medium renewal period).

In the control and at the loading rate of 100 mg/L (mean measured concentration: 38 µg/L) no significant mortality or other visible abnormalities were determined at the test fish during the test period of 96 hours.

The summary of the biological results is as follows:

– 96-hour LL50 / LC50: Loading rate >100 mg/L and mean measured concentration >38 µg/L

– 96-hour LL0 / LC0: Loading rate ≥100 mg/L and mean measured concentration ≥38 µg/L

– 96-hour LL100 / LC100: Loading rate >100 mg/L and mean measured concentration > 38 µg/L

– 96-hour NOELR / NOEC: Loading rate ≥100 mg/L and mean measured concentration ≥38 mg/L µg/L

– 96-hour LOELR / LOEC: Loading rate >100 mg/L and mean measured concentration >38 µg/L

Based on the findings of the study, the test item FAT #: 93580/A had no acute toxic effects on zebra fish up to its solubility limit in test water under the present test conditions.

Description of key information

The study describes acute toxicity of the test item FAT #: 93580/A to Zebra fish (Danio rerio) was determined in a 96 hour semi-static test with daily test medium renewal according to OECD TG 203.

In the control and at the loading rate of 100 mg/L (mean measured concentration: 38 µg/L) no mortality or other visible abnormalities were noted in the test fish during the test period of 96 hours.

The summary of the biological results is as follows:

– 96-hour LL50 / LC50: Loading rate >100 mg/L and mean measured concentration >38 µg/L

– 96-hour LL0 / LC0: Loading rate ≥100 mg/L and mean measured concentration ≥38 µg/L

– 96-hour LL100 / LC100: Loading rate >100 mg/L and mean measured concentration >38 µg/L

– 96-hour NOELR / NOEC: Loading rate ≥100 mg/L and mean measured concentration ≥38 mg/L µg/L

– 96-hour LOELR / LOEC: Loading rate >100 mg/L and mean measured concentration >38 µg/L

Based on the findings of the expxeriment, the test item FAT #: 93580/A had no acute toxic effects on zebra fish up to its solubility limit in test water under the present test conditions.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L