Sebacohydrazide

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Oct 2018 - 31 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sebacohydrazide

EC Number:

213-126-8

EC Name:

Sebacohydrazide

Cas Number:

925-83-7

Molecular formula:

C10H22N4O2

IUPAC Name:

sebacohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Test item name (as stated in report): SDH
Test item storage: at room temperature protected from light

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations and the control
- Sampling method: 2.8 mL from the approximate centre of the test vessels, at t=0 h and t=48 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to test medium. Test solutions were prepared at 99 mg/L in 200 mL test medium with a 15-minute period of ultrasonic treatment to accelerate dissolution of test item in test medium. Thereafter, another 800 mL of test medium was added to reach a final volume of 1000 mL. Subsequently, magnetic stirring was applied until the test item was completely dissolved in test medium (i.e. approximately 2¼ - 2½ hours). Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives
- Other relevant information: A correction factor of 1.06 was applied during the experimental phase of the study. Based on the purity information that became available later (93.4% purity), all further concentrations were based on the pure test item using a correction factor of 1.07. The change of the correction factor from 1.06 to 1.07 was judged not to impact the study integrity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Age at study initiation: <24 hours
- Source: In-house laboratory culture with a known history
- Method of breeding: cyclical parthenogenesis under specified breeding conditions
- Age of parental stock: >2 weeks
- Feeding during test: no
- Validity of batch: 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.

CULTIVATION
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L (expressed as CaCO3)

Test temperature:

19-20°C

pH:

At t=0 h: 7.8
At t=48 h: 8.0

Dissolved oxygen:

At t=0 h: 8.5-8.7 mg/L
At t=48 h: 8.9-9.1 mg/L

Nominal and measured concentrations:

Nominal concentrations: 9.9, 18, 32, 55 and 99 mg/L
Measured concentrations: 10, 19, 33, 55 and 103 mg/L at start of the test (99-106% relative to nominal). During the exposure period, the concentrations remained stable and were measured to be at 99 – 102% relative to nominal concentrations at the end of the test. Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations. See Table 1 in 'Any other information on results' for more details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, with 50 mL test solution
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: Within 40 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium (prepared with water purified by reverse osmosis)
- Culture medium different from test medium: yes, M7
- Intervals of water quality measurement: Dissolved oxygen and pH: at beginning and end of the test, for all concentrations and the control. Temperature: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): At 24 hours and at 48 hours.

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: 0.099, 0.99, 9.9, 99 mg/L
- Results used to determine the conditions for the definitive study: yes, the expected EC50 was between nominally 9.9 and 99 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, performed Nov 2018.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 45-60 mg/L

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

99 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Obtained from experimental observations on immobility without the use of any statistical analysis.

Details on results:

- Behavioural abnormalities: none observed in the Control group.
- Mortality of control: none
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
- No immobility was observed in the control and at the three lowest test concentrations throughout the test, while 70 and 95% immobility was observed at 55 and 99 mg/L, respectively, at the end of the test. After 24 hours of exposure, 50% immobility was observed at the highest test concentration of nominally 99 mg/L.
- Small responses at the retention time of the test item were detected in the chromatograms of the blank QC samples. It was considered to derive from carry-over in the analytical system since similar responses were found in the analytical blanks. This did not impact the quantification in the concentration range of 0.005 – 99 mg/L.

Results with reference substance (positive control):

The 48h-EC50 was 0.40 mg/L with a 95% confidence interval between 0.33 and 0.48 mg/L.
The actual responses in this reference test with K2Cr2O7 are generally within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.28 and 0.90 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Reported statistics and error estimates:

The 48h-EC50 was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations using the maximum likelihood estimation method.
The 24h-EC50 could not be determined using any regression method due to the lack of fit with experimental data. Instead, the 24h-EC50 was obtained from experimental observations on immobility without the use of any statistical analysis.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hour]	Date of sampling	Date of analysis (a)	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	23 Oct 2018	30 Oct 2018	0	0.000017 (b)	n.a.
			9.9	10.2	103
			18	19.0	106
			32	33.1	103
			55	54.6	99
			99	103	104
48	25 Oct 2018	30 Oct 2018	0	0.0000018 (b)	n.a.	n.a.
			9.9	10.1	102	99
			18	18.3	102	96
			32	32.1	100	97
			55	54.5	99	100
			99	97.7	99	95

(a) Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(b) Estimated value, calculated by extrapolation of the calibration curve. This is probably caused by carry over since a similar response was observed in the analytical blank.

n.a. Not applicable.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test 

Time (h)	Replicate	Test item Nominal conc. (mg/L)
		Control	9.9	18	32	55	99
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	0	1
	B	0	0	0	0	0	4
	C	0	0	0	0	0	2
	D	0	0	0	0	0	3
	Total immobilised	0	0	0	0	0	10
	Effect %	0	0	0	0	0	50
48	A	0	0	0	0	5	4
	B	0	0	0	0	2	5
	C	0	0	0	0	4	5
	D	0	0	0	0	3	5
	Total immobilised	0	0	0	0	14	19
	Effect %	0	0	0	0	70	95

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For details on validity criteria, please refer to section 'Overall remarks'

Conclusions:

48h-EC50 for Daphnia magna exposed to the test item was 52 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 45 and 60 mg/L).

Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to the test item at nominal concentrations of 9.9, 18, 32, 55 and 99 mg/L and an untreated control (all with 4 replicates per concentration and 5 daphnids per replicate). Measured concentrations were at 99 -106% relative to nominal concnetrations throughout the test. Therefore, effect parameters were based on nominal concentrations.

No immobility was observed in the control and at the three lowest test concentrations throughout the test, while 70 and 95% immobility was observed at 55 and 99 mg/L at the end of the test.

The 48h-EC50 for Daphnia magna exposed to the test item was 52 mg/L (95% confidence interval between 45 and 60 mg/L), based on analytically confirmed nominal concentrations. The study met all validity criteria and is considered to be reliable without restrictions.