Isophthalohydrazide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Mar 2018 - 25 Apr 2018

Reliability:

1 (reliable without restriction)

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006; Annex 5 corrected 2011

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: all test concentrations and control
- Sampling method: 2.0 mL from the approximate centre of the test vessels, at t=0 h, t=24 h and t=72 h
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration (i.e. 10 mg/L) but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to the test medium. Preparation of test solutions started with the highest concentration of 100 mg/L applying approximately 10-20 minutes of magnetic stirring to completely dissolve 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.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

- Controls: Test medium without test item or other additives

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture.
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis)
- Pre-culture medium and test medium: M2, according to OECD TG201

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21 - 23°C

pH:

At t=0 h: 8.2-8.3
At t=72 h: 7.9-8.1

Nominal and measured concentrations:

Nominal: 0.32, 1.0, 3.2, 10, 32, 100 mg/L
Measured: at start: 0.083, 0.37, 1.6, 7.6, 30 and 101 mg/L, at 72 h: Effect parameters were based on Time Weighted Average concentrations: 0.028, 0.056, 0.44, 3.3, 20, 84 mg/L (see 'Any other information on materials and methods' for details on the TWA calculation)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 285 x 10^4 cells/mL
- 3 replicates of each test concentration
- 6 replicates of the control
- 1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
- 1 or 2 replicates of each test concentration without algae.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water (tapwater purified by reverse osmosis)
- Intervals of water quality measurement: pH: at the beginning at at the end of the test. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 80 to 82 µE.m^-2.s^-1.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Appearance of the cells: At the end of the final test microscopic observations were performed on the test item concentration of nominally 32 mg/L to observe for any abnormal appearance of the algae compared to the control.

TEST CONCENTRATIONS
- Combined Limit/Range finding study test concentrations: 0.10, 1.0, 10, 100 mg/L
- 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

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 84 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

8.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 4.4-12 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Details on results:

- Exponential growth in the control (for algal test): yes
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 20 mg/L when compared to the control.
- A dose-related increase of inhibition of growth rate was observed at all test concentrations, up to 36% inhibition, at the end of the test. Statistically significant inhibition of growth rate was found at the three highest test concentrations. However, the effects on growth rates at TWA concentration of 3.3 mg/L was considered biologically not relevant; i.e. was below 10%. Therefore, the NOEC for growth rate inhibition based on biological relevance was set to 3.3 mg/L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- The EC50 for growth rate inhibition (72h-ErC50) was 1.6 mg/L with a 95% confidence interval ranging from 1.5 to 1.6 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Determination of NOEC: Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller.
Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition versus the logarithms of the corresponding TWA concentrations.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hours]	Date of sampling	Date of analysis (a)	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0	16 Apr 2018	25 Apr 2018	0	n.d.	n.a.
			0.32	0.0831	26
			1.0	0.373	37
			3.2	1.61	50
			10	7.55	76
			10 (b)	7.63	76
			32	30.1	94
			100	101	101
24	17 Apr 2018	25 Apr 2018	0	n.d.	n.a.	n.a.
			0.32	0.058 (c)	18	70
			1.0	0.061 (c)	6.1	16
			3.2	0.594	19	37
			10	4.27	43	57
			10 (b)	4.64	46	61
			32	23.0	72	76
			100	92.1	92	91
72	19 Apr 2018	25 Apr 2018	0	n.d.	n.a.	n.a.
			0.32	n.d.	n.a.	n.a.
			1.0	<LOD	n.a.	n.a.
			3.2	0.047 (c)	1.5	2.9
			10	0.984	10	13
			10 (b)	1.54	15	20
			32	12.0	37	40
			100	66.9	67	67

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

(b) Without algae.

(c) Estimated value, calculated by extrapolation of the calibration curve.

LOD The limit of detection of the analytical method was determined to be 0.0022 mg/L.

n.d. Not detected.

n.a. Not applicable.

Table 2: Growth Rate And Percentage Inhibition For The Total Test Period

TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.883	0.0235	6
0.028	1.879	0.0184	3	0.23
0.056	1.858	0.0259	3	1.3
0.44	1.866	0.0017	3	0.92
3.3	1.781	0.0509	3	5.4^#
20	1.558	0.0067	3	17*
84	1.211	0.1531	3	36*

* - effect was statistically significant

^#- effect was statistically significant but biologically not relevant (<10%)

Table 3: Growth Rate And Percentage Inhibition At Different Time Intervals

TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.444		1.752		1.453
0.028	3	2.411	1.4	1.796	-2.5	1.429	1.6
0.056	3	2.383	2.5	1.753	-0.056	1.439	1.0
0.44	3	2.463	-0.77	1.630	7.0	1.504	-3.5
3.3	3	2.394	2.0	1.453	17	1.496	-2.9
20	3	2.428	0.67	1.160	34	1.086	25
84	3	2.289	6.3	0.751	57	0.593	59

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The EC50 for growth rate inhibition (72h-ERC50) was beyond the range of concentrations tested, i.e. exceeded a TWA concentration of 84 mg/L.
The 72h-EC10 for growth rate inhibition was at a TWA concentration of 8.1 mg/L.
The 72h-NOEC for growth rate inhibition was at a TWA concentration of 0.44 mg/L based on statistical significance.
The 72h-NOEC for growth rate inhibition was at a TWA concentration of 3.3 mg/L based on biological relevance.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test item at Time Weighted Average (TWA) concentrations of 0.028, 0.056, 0.44, 3.3, 20 and 84 mg/L (3 replicates per concentration) and an untreated control (6 replicates). Effect parameters were based on TWA concentrations. Growth rates of the algae exposed to the test item were increasingly reduced with increasing concentrations, resulting in 36% inhibition at the highest test concentration. The EC50 for growth rate inhibition (72h-ErC50) was >84 mg/L, 72h-ErC10 was 8.1 mg/L. The 72h-NOErC was 0.44 mg/L and 3.3 mg/L, based on statistical significance and biological relevance, respectively. The study met all validity criteria, and is considered reliable without restriction.