Isophthalohydrazide

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:

12 Mar 2018 - 18 May 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

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Isophthalohydrazide

EC Number:

220-425-7

EC Name:

Isophthalohydrazide

Cas Number:

2760-98-7

Molecular formula:

C8H10N4O2

IUPAC Name:

isophthalohydrazide

Test material form:

solid: particulate/powder

Details on test material:

Appearance: White crystal powder
Test item storage: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test concentrations
- Sampling method: 2.0 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: Preparation of test solutions started with the highest concentration of 100 mg/L applying from 10 to 35 minutes of magnetic stirring to accelerate dissolution of the test item in medium. 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.
- Evidence of undissolved material: The saturated solution prepared for the combined limit/range-finding test was observed to contain undissolved material, which was filtered out using a 0.45 µm membrane filter (RC55, Whatman). It was thus concluded that any undissolved material was not test item related. The solutions for the limit and final tests did not contain any undissolved material.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: >2 weeks old
- Age at test initiation: <24 hours
- 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 as CaCO3

Test temperature:

20 - 21°C

pH:

At start: 7.8
At end (t=48 h): 8.1

Dissolved oxygen:

At start: 8.4 - 9.0 mg/L
At end (t=48 h): 8.9 - 9.0 mg/L

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56, 100 mg/L
Measured concentrations were at the level of nominal throughout the test duration (i.e., 91-103% of nominal concentrations). Based on these results, effects parameters were based on the analytically confirmed nominal concentrations. See Table 1 in 'Any other information on results' for details on measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL all-glass with a fill volume of 50 mL
- Aeration: no
- Feeding: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, prepared with tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes, M7 medium
- Intervals of water quality measurement: Dissolved oxygen and pH: at beginning and end of the test, for all test 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 photoperiod daily

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

RANGE-FINDING STUDY
A Combined Limit/Range-Finding test and a Limit test were performed prior to the Final test.
- Test concentrations: 0.1, 1.0, 10 and 100% of a saturated solution prepared at 100 mg/L (Combined Limit/Range-Finding test); 0 and 100 mg/L (Limit test).
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, performed March 2018

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

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.

Immobility at the highest test concentration was observed in all three tests performed (i.e. 25, 70 and 45% immobility in the combined limit/range-finding test, limit test and final test, respectively, at the end of the test), however, only in the limit test the effect was >50%. The observed difference could be due to a difference in sensitivity of the daphnids. Additionally, no toxicity was observed after 24 hours of exposure in any of the tests performed and, in both the combined limit/range-finding test and final test, a high variability in immobility was observed between vessels. This could imply a slightly delay in toxicity around the 48 hours of exposure.
In conclusion, it was decided to base the effects parameters on the results of two out of the three tests, i.e. the combined limit/range-finding test and the final test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The 24h-EC50 was 0.80 mg/L with a 95% confidence interval between 0.71 and 0.90 mg/L.
- The 48h-EC50 was 0.33 mg/L with a 95% confidence interval between 0.29 and 0.38 mg/L.
- The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis #	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	07 May 2018	18 May 2018	0	n.d.	n.a.
			10	9.36	94
			18	17.8	99
			32	31.2	97
			56	57.5	103
			100	102	102
48	09 May 2018	18 May 2018	0	n.d.	n.a.	n.a.
			10	9.08	91	97
			18	17.5	97	98
			32	30.9	96	99
			56	56.4	101	98
			100	102	102	99

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

n.d. Not detected.

n.a. Not applicable.

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

Time (h)	Replicate	Measured concentration (mg/L)
		Control	10	18	32	56	100
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	0
	B	0	0	0	0	0	0
	C	0	0	0	0	0	0
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0	0	0	0	4
	B	0	0	0	0	0	3
	C	0	0	0	0	0	2
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	9
	Effect %	0	0	0	0	0	45

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

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

Conclusions:

The 48h-EC50 for Daphnia magna exposed to the test substance was > 100 mg/L based on analytically confirmed nominal concentrations.

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 10, 18, 32, 56 and 100 mg/L and an untreated control (4 replicates, 5 daphnids per replicate). Measured concentrations were at 91 -103% relative to nominal throughout the test. Therefore, effect parameters were based on nominal concentrations. No immobility was observed in the control and the four lowest test concentrations throughout the test, while 45% effect was observed at the highest concentration after 48 h of exposure. The 48h-EC50 for Daphnia magna exposed to the test item exceeded a nominal concentration of 100 mg/L. The study is considered to be reliable without restrictions.