Fatty acids, C18-unsatd., phosphates

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:

The study will be available 27/06/2018 (A month after Unaudited Draft Report is delivered which will be provided on 27 May 2018)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures

Version / remarks:

OECD series on testing and assessment number 23, 2000.

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 02 November 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glass wool containing the undissolved residue was kept for possible analysis.

Frequency at t=0 h, t=24h and t=96h
Volume 2.7 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Additionally, reserve samples of 2.7 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Details on test solutions:

The batch of Fatty acids, C18-unsatd., phosphates tested was a yellow liquid UVCB substance which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. Glassware used for the preparation of test solutions and exposure phase was silanized to prevent adsorption of test item to the vessels.
Preparation of test solutions started with loading rates individually prepared in the range of 1.0 and 100 mg/L. A two-day period of magnetic stirring was applied to accelerate dissolution of the test item in test medium. The obtained mixtures were allowed to settle overnight. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure, except for the WAF prepared at 100 mg/L used in the full final test which was observed to be slightly hazy. Any residual volumes were discarded.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species: arp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source:Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.

Mean length: Range-finding test: 2.3 ± 0.1 cm
Limit test: 2.9 ± 0.2 cm
Full test: 2.9 ± 0.2 cm
Mean weight: Range-finding test: 0.20 ± 0.04 g
Limit test: 0.25 ± 0.07 g
Full test: 0.26 ± 0.09 g

Characteristics: F1 from a single parent-pair bred in UV-treated water.
Reason for selection: This system has been selected as an internationally accepted species.
Total fish used:65

Test type:

static

Water media type:

freshwater

Remarks:

Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)

Limit test:

yes

Total exposure duration:

96 h

Hardness:

180 mg CaCO3

Test temperature:

20.5 ± 0-.5

pH:

7.7 ± 0.3

Dissolved oxygen:

8.6 ± 1

Nominal and measured concentrations:

WAFs prepared at Loading rate (mg/L): 4.6, 10, 22, 46 and 100.

The test item concentrations based on constituent m/z 391.3 measured in the WAF prepared at the loading rate of 100 mg/L were 46, 81 and 39 mg/L at the beginning of range-finding, limit and full test, respectively. This corresponds better to the differences in effects observed in respective tests however does not provides full explanation of the observed differences. Therefore, it is assumed that differences in the level of dissolutions of some poorly soluble components could be responsible for the observed differences in mortality. As the concentration measured based on constituent m/z 391.3 represent worst-case (poorly soluble component), it is justified to base the effect parameters on concentrations determined based on this constituent.

Details on test conditions:

Test duration:96 hours
Test type:Static
Test vessels:3.2 litres, silanized all-glass, containing 3 litres of test solution.
Test medium: Adjusted ISO medium with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
Number of fish: 7 per test group
Loading: 0.61 g fish/Litre, i.e. 7 fish per 3 litres of test medium
Illumination: 16 hours photoperiod daily
Aeration: Aeration was introduced after approximately 48 hours of exposure.
Feeding: No feeding from 24 hours prior to the test and during the total test period
Introduction of fish: 13 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 1.0 unit and 1.0°C.

Reference substance (positive control):

yes

Remarks:

The reference item was pentachlorophenol (PCP, SIGMA, Art. P2604, Batch MKBW8316V)

Key result

Duration:

96 d

Dose descriptor:

LC50

Effect conc.:

16.137 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence interval between 12 and 21 mg/L

Details on results:

Range-Finding test
The results of the range-finding test are presented in Table 2. No mortality or clinical effects were observed at any of the test concentrations during the test period. Following these results, samples taken from the WAF prepared at 100 mg/L were analysed employing two out of several peaks observed in the chromatograms of the test item. Based on the peak at m/z 151.0, the concentration at the start of the test was 97 mg/L and remained stable throughout the test, i.e. was at 105% of the initial concentration at the end of the test.. Based on the peak at m/z 391.3, the concentration at the start of the test was 46 mg/L, which decreased to 66% of the initial concentration at the end of the test (see also Table 4 of the appended Analytical Report).
All test conditions were maintained within the limits prescribed by the study plan.

Limit Test
The results of the limit test are presented in Table 3. No mortality or clinical effects were observed in the control, while 100% mortality was observed at the limit concentration after 24 hours of exposure.
Following these results, samples taken from the WAF prepared at 100 mg/L were analysed.
Based on the peak at m/z 151.0, the concentration at the start of the test was 106 mg/L and remained stable throughout the test, i.e. was at 100% of the initial concentration at the end of the test (see also Table 5 of the appended Analytical Report).
Based on the peak at m/z 391.3, the concentration at the start of the test was 81 mg/L, which decreased to 13% of the initial concentration at the end of the test (see also Table 6 of the appended Analytical Report).
After 24 hours of exposure, white particulate material was observed floating in the solution prepared at the limit concentration. During the following 24 hours, the same solution turned hazy. The source of the undissolved material and hazy appearance is unknown and could probably be caused by precipitation of test item and/or components of the test medium.
All test conditions were maintained within the limits prescribed by the study plan.

Final Test
Mortality observed at the limit test was contradictory to the results obtained during the range-finding test. Hence, an additional full final test was performed.

Measured Concentrations
Samples taken from all test concentrations were analysed. Based on the peak at m/z 151.0, the concentrations at the start of the test were 5.9, 14, 30, 58 and 103 mg/L in WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L, respectively. The measured concentrations were at 99-126% of the initial concentrations at the end of the test.
Based on the peak at m/z 391.3, the concentrations at the start of the test were 0.56, 3.0, 3.6, 17 and 39 mg/L in WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L, respectively. At the end of the test, no test item concentrations could be detected in the three lowest test concentrations. In the WAFs prepared at loading rates of 46 and 100 mg/L, concentrations of 2.4 and 3.6 mg/L were measured, respectively, at the end of the test.
Following the worst-case principle, the effect parameters were based on the results obtained for the peak at m/z 391.3. To this end, the Time Weighted Average (TWA) concentrations were calculated for the peak at m/z 391.3 and used to determine the effect parameters. Since constituent m/z 391.3 could not be measured in the three lowest test concentrations, the TWA concentrations were only calculated for the WAFs prepared at loading rates 46 and 100 mg/L (see Table 4). These two concentrations were also only two relevant for the calculations of LCx values.

Mortality and Other Effects
Table 5 shows the mortality data recorded during the final test. No mortality or other clinical effects were observed in the control and at the four lowest test concentrations throughout the test. In the highest WAF, 43% of fish survived the 96 hour exposure period. After 24 and 48 hours of exposure, the surviving fish exposed to the highest test concentration were observed to swim close to the surface (see Table 5).
The responses recorded in this test allowed for reliable determination of an LC50.

Determination of Effect Concentrations
Table 6 shows the effect parameters based on TWA concentrations.

Experimental Conditions
The results of measurement of pH and dissolved oxygen concentrations are presented in Table 7. Aeration was introduced after approximately 48 hours of exposure, as the oxygen concentration had dropped below the optimum level for testing with carp, i.e. below 5 mg/L, in some test vessels. The temperatures measured during the study in the various test vessels are presented in Table 8. Generally, all test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Sublethal observations / clinical signs:

Table 2. Incidence of Mortality and Total Mortality during the Range-Finding Test

Test item^1 Loading rate (mg/L)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		4h	24h	48h	72h	96h
1.0	3	0	0	0	0	0	0
10	3	0	0	0	0	0	0
100	3	0	0*	0	0	0	0

¹Fatty acids, C18-unsatd., phosphates
* Fish were observed to swim closer to the bottom of the test vessel compared to the other treatments

Table 3. Incidence of Mortality and Total Mortality during the Limit Test

Test item^1 Loading rate (mg/L)	Initial number of fish	Cumulative mortality					Total Mortality (%)
		2h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
100	7	0	7	7	7	7	100

¹Fatty acids, C18-unsatd., phosphates

Table 4. Measured Concentrations Versus Nominal Concentrations (m/z391.3)

Test item^1 Loading rate (mg/L)	Measured concentrations (mg/L)			TWA(mg/L)
	t=0h	t=24h	t=96h
46	17.0	13.9	2.40	8.2
100	38.8	33.8	3.56	17

¹Fatty acids, C18-unsatd., phosphates

Table 5. Incidence of Mortality and Total Mortality during the Final Test

Test item^1 Loading rate (mg/L)	Initial number of fish	Cumulativemortality					Total Mortality (%)
		3h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
4.6	7	0	0	0	0	0	0
10	7	0	0	0	0	0	0
22	7	0	0	0	0	0	0
46	7	0	0	0	0	0	0
100	7	0	3 (4)	4 (1)	4	4	57

¹Fatty acids, C18-unsatd., phosphates; ( ) – Number of fish observed to swim close to the surface.

Table 6. Effect Parameters

Parameter	Fatty acids, C18-unsatd., phosphates TWA conc. (mg/L)	95%-confidence interval (mg/L)
24h-LC50	>17
48h-, 72h- and 96h-LC50	16	12-21

Table 7. pH-Values and Dissolved Oxygen Concentrations (mg/L) during the Final Test

Test item^1 Loading rate (mg/L)	Day 0		Day 1		Day 2		Day 3		Day 4
	pH^2	O2	pH	O2	pH	O2	pH	O2	pH	O2
Control	7.8	8.5	8.0	7.2	8.0	6.4	7.9	8.3	8.0	8.8
4.6	7.9	8.6	7.9	7.1	7.9	6.3	7.9	8.5	8.0	8.9
10	7.7	8.6	7.7	7.2	7.5	4.7	7.9	8.6	8.0	9.2
22	7.6	8.5	7.6	7.2	7.3	5.3	7.8	8.5	8.0	9.2
46	7.3	8.1	7.6	7.1	7.3	6.4	7.8	8.5	7.9	9.3
100	7.2	7.6	7.5	7.0	7.3	5.3	7.8	8.4	7.9	9.6

¹Fatty acids, C18-unsatd., phosphates;²pH of culture medium was: 7.4

 

 

Table 8. Temperatures (°C) measured during the Final Test

Test item^1 Loading rate (mg/L)	Day 0^2	Day 1	Day 2	Day 3	Day 4
Control	22	21	21	21	21
4.6	21	21	21	21	21
10	21	21	22	21	21
22	21	21	22	21	21
46	21	21	22	21	21
100	21	21	22	21	21

¹Fatty acids, C18-unsatd., phosphates;²Temperature of culture medium was: 22°C.

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 was 16 mg/L based on TWA exposure concentrations (95% confidence interval between 12 and 21 mg/L) and was already reached after 48 hours of exposure.

Executive summary:

The objective of the study was to evaluate Fatty acids, C18-unsatd., phosphates for its ability to generate acute toxic effects inCyprinus carpioduring an exposure period of 96 hours and, if possible, to determine the LC₅₀at all observation times. The study procedure described in this report was based onthe OECD guideline No. 203, 1992. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

The batch of Fatty acids, C18-unsatd., phosphates tested was a yellow liquid UVCB substance and not completely soluble in test medium at the loading rates initially prepared. Due to the observed degree of adsorption during the analytical method development, all glassware employed in this study was silanized prior to use. Water Accommodated Fractions (WAFs) were individually prepared and used as test concentrations.

A final test was performed based on the results of preceding range-finding and a limit tests. Seven fish per group were exposed to an untreated control and WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L.The total exposure period was 96 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 hours of exposure and at the end of the test.

No mortality or other clinical effects were observed in the control and at the four lowest test concentrations throughout the test, while 57% of fish exposed to the highest test concentration did not survive the 96-hour exposure period.

Samples taken from all test concentrations were analysed employing two out of several peaks observed in the chromatograms of the test item. Based on the peak atm/z151.0, the concentrations at the start of the test were 5.9, 14, 30, 58 and 103 mg/L in WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L, respectively. The measured concentrations were at 99-126% of the initial concentrations at the end of the test. Based on the peak atm/z391.3, the concentrations at the start of the test were 0.56, 3.0, 3.6, 17 and 39 mg/L in WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/L, respectively. At the end of the test, no test item concentrations could be detected in the three lowest test concentrations. In the WAFs prepared at loading rates of 46 and 100 mg/L, concentrations of 2.4 and 3.6 mg/L were measured at the end of the test, respectively. Following the worst-case principle, the effect parameters were based on the results obtained for the peak atm/z391.3. The Time Weighted Average (TWA) concentrations calculatedfor the relevant WAFs were 8.2 and 17 mg/L at the WAFs prepared at 46 and 100 mg/L, respectively. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion,the 96h-LC₅₀was 16 mg/L based on TWA exposure concentrations (95% confidence interval between 12 and 21 mg/L) and was already reached after 48 hours of exposure.

Description of key information

The acute test in Carp (Cyprinus carpio) to Fatty acids, C-18 -unsatd., phosphates was determined using static test system and it was performed based on the results of preceding range-finding and a limit test. Seven fish per group were exposed to an untreated control and WAFs prepared at loading rates of 4.6, 10, 22, 46 and 100 mg/l and 100 mg/l in the limit test.

The study was conducted according to OECD 203 and followed OECD Principles of Good Laboratory Practice (GLP). No unusual observations or deviations from standard test protocol were noted. As a result, the 96-h LC50 measured was 16 mg/L.

Based on the available data, the 96-h LC50-value was selected as a key value.

The data obtained from testing on freshwater species has been used as basis for assessment of effects in marine environment as well as for extrapolation of the measured effects to other compartments within the aquatic ecosystem (e.g. sediment) and soil.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

16 mg/L

Marine water fish

Marine water fish

Effect concentration:

16 mg/L