Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)

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:

21 July 2018 to 26 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Frequency: at t=0 h and t=48 h
- Volume: 2.0 mL from the approximate centre of the test vessels
- Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
At the end of the exposure period, the replicates were not pooled at each concentration before sampling. Instead, samples were taken from one replicate per test concentration.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The batch of the test material used was an amber 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 material.
- Preparation of test solutions started with loading rates individually prepared at 1.0, 10 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test material in medium. The obtained mixtures were allowed to settle for approximately one day. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. At the end of the preparation procedure, the test solutions prepared at the two lowest test concentrations were clear and colourless, while the test solution prepared at the limit concentration was slightly hazy and yellow. The highest test concentration in the final test was observed with a laser pen to check for the presence of Tyndall effect. No undissolved particles were observed.
- Any residual volumes were discarded.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Daphnia magna (Crustacea, Cladocera) (Straus, 1820) at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions
- Source: In-house laboratory culture with a known history
- Age: less than 24 hours old, from parental daphnids of more than two weeks old
- Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20 %, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Feeding during test: no

BREEDING
- Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
- Temperature of medium: 18-22 °C
- Feeding: Daily, a suspension of fresh water algae.
- Culture medium: M7, as prescribed by Dr. Elendt-Schneider. The following salts and vitamins were added to freshly prepared test medium to reach the following concentrations: Salts: H3BO3 0.73 mg/L, FeSO4.7H2O 0.25 mg/L, MnCl2.4H2O 0.092 mg/L, LiCl 0.078 mg/L, RbCl 0.018 mg/L, SrCl2.6H2O 0.039 mg/L, Na2MoO4.2H2O 0.016 mg/L, NaBr 0.0041 mg/L, CuCl2.2H2O 0.0043 mg/L, ZnCl2 0.013 mg/L, CoCl2.6H2O 0.010 mg/L, KI 0.0033 mg/L, Na2SeO3 0.0022 mg/L, NH4VO3 0.00059 mg/L, Na2EDTA.2H2O 0.64 mg/L, Na2SiO3.5H2O 7.6 mg/L, NaNO3 0.28 mg/L, KH2PO4 0.15 mg/L and K2HPO4 0.19 mg/L, Vitamins: Thiamine hydrochloride 75.0 μg/L, B12 1.0 μg/L and Biotin 0.75 μg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L (as CaCO3)

Test temperature:

20-22 °C

pH:

8.1-8.2

Dissolved oxygen:

7.9-8.9 mg/L

Nominal and measured concentrations:

Nominal: WAFs individually prepared at loading rates of 1.0, 10 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Fill volume: 50 mL
- Aeration: No aeration of the test solutions was applied
- No. of organisms per vessel: 5
- Daphnids were introduced to the vessels within 28 minutes after preparation of the test solutions.
- No. of vessels per concentration (replicates): 5 for limit concentration and 2 for lower concentrations
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water): CaCl2.2H2O 211.5 mg/L, MgSO4.7H2O 88.8 mg/L, NaHCO3 46.7 mg/L and KCl 4.2 mg/L. The hardness of test medium expressed as CaCO3: 180 mg/L with a pH between 6 and 9.
- Intervals of water quality measurement: pH and dissolved oxygen were recorded at the beginning and at the end of the test, for the limit concentration and the control. The temperature of the medium was continuously measured in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A daily photoperiod of 16 hours.

EFFECT PARAMETERS MEASURED
- Immobility (including mortality) was recorded at 24 hours and at 48 hours.

INTERPRETATION
Acceptability of the Test
- In the control, no daphnids became immobilised or showed other signs of disease or stress, for example discolouration or unusual behaviour such as trapping at the surface of the medium.
- The dissolved oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Data Handling
- The average exposure concentrations were calculated as √(Ct=0 x Ct=48), being the geometric means of the concentrations of the test material measured in the samples taken at the start (Ct=0) and the end of the test (Ct=48).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

MEASURED CONCENTRATIONS
- Samples taken from all test concentrations and the control were analysed. At the start of the test, the measured concentration at the highest WAF tested was 7.7 mg/L, which decreased below the Limit of Quantification (LOQ) at the end of the test and was estimated to be 0.0020 mg/L by extrapolation of the calibration curve. No clear test material related responses could be measured at the lower WAFs tested already at the start of the test. It should be noted that at the highest test solution, a floating layer of undissolved material was observed in all the test vessels throughout the test. This was supposedly caused by oversaturation.
- Based on these results, effect parameters were based on the average exposure concentration at the limit concentration, calculated to be 0.12 mg/L.

IMMOBILITY
- No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

Reported statistics and error estimates:

No statistical analysis was performed.

Table 1: Number of Introduced Daphnids and Incidence of Immobility

Time (h)	Replicate	Test Material Loading Rate (mg/L)
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total Introduced	20	10	10	20
24	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total Immobilised Effect (%)	0 0	0 0	0 0	0 0
48	A	0	0	0	0
	B	0	0	0	0
	C	0			0
	D	0			0
	Total Immobilised Effect (%)	0 0	0 0	0 0	0 0

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the 48h-EC50 for Daphnia magna exposed to the test material was beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.12 mg/L being considered as the maximum soluble concentration of test material in test medium.

Executive summary:

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions.

The objective of the study was to evaluate the test material for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

The batch of the test material tested was an amber liquid UVCB substance which was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition, ten daphnids per group (5 per replicate, duplicate) were exposed WAFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed. At the start of the test, the measured concentration at the highest WAF tested was 7.7 mg/L, which decreased below the Limit of Quantification (LOQ) at the end of the test and was estimated to be 0.0020 mg/L by extrapolation of the calibration curve. No clear test material related responses could be measured at the lower WAFs tested already at the start of the test. Based on these results, effect parameters were based on the average exposure concentration at the limit concentration, calculated to be 0.12 mg/L.

No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

Under the conditions of this study, the 48h-EC50 for Daphnia magna exposed to the test material was beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.12 mg/L being considered as the maximum soluble concentration of test material in test medium.

Description of key information

Under the conditions of the study, the 48h-EC50 for Daphnia magna exposed to the test material was beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.12 mg/L being considered as the maximum soluble concentration of test material in test medium.

Additional information

The short-term toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guideline OECD 202, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to evaluate the test material for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

The batch of the test material tested was an amber liquid UVCB substance which was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WAF prepared at a loading rate of 100 mg/L, in a limit test. In addition, ten daphnids per group (5 per replicate, duplicate) were exposed WAFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Samples taken from all test concentrations and the control were analysed. At the start of the test, the measured concentration at the highest WAF tested was 7.7 mg/L, which decreased below the Limit of Quantification (LOQ) at the end of the test and was estimated to be 0.0020 mg/L by extrapolation of the calibration curve. No clear test material related responses could be measured at the lower WAFs tested already at the start of the test. Based on these results, effect parameters were based on the average exposure concentration at the limit concentration, calculated to be 0.12 mg/L.

No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

Under the conditions of the study, the 48h-EC50 for Daphnia magna exposed to the test material was beyond the range of concentrations tested, i.e. exceeded an average exposure concentration of 0.12 mg/L being considered as the maximum soluble concentration of test material in test medium.