Sebacohydrazide

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:

01 Oct 2018 - 03 Nov 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 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:

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, t=24 h and t=72 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 with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at nominally 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 test medium. Preparation of test solutions started with the highest concentration of 99 mg/L. Test item was added to 200 mL test medium after which the 15 minutes of ultrasonic treatment were applied. Thereafter, another 800 mL of test medium was added to reach a final volume of 1000 mL. Subsequently, two hours of magnetic stirring was sufficient to completely dissolve the test item in test 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.
- 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):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 (M1) 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 (M2) 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).
- Pre-culture medium and test medium: M2, according to OECD 201.

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:

23 - 24°C

pH:

At t=0 h: 8.1 - 8.3
At t=72 h: 7.6 - 7.7

Nominal and measured concentrations:

Nominal concentrations: 0.99, 3.2, 9.9, 32, 99 mg/L
Initial measured concentrations: 0.57, 2.1, 8.0, 27, and 89 mg/L at nominal concentrations of 0.99, 3.2, 9.9, 32 and 99 mg/L, respectively.
Measured concentrations at 72 h decreased to 9.2 – 63% of initially measured concentrations. Time Weighted Average Concentrations were calculated, see 'Any other information on materials and methods' for the calculation and see 'Any other information on results' for details on measured and TWA concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 210 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 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 OECD 201, formulated using water purified by reverse osmosis.
- Intervals of water quality measurement: pH: at beginning and end of test; temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod and light intensity: Continuously using TLD-lamps with a light intensity within the range of 89 to 93 µE.m-2.s-1.
- Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

EFFECT PARAMETERS MEASURED
- Cell density was measured at t=24 h, t=49 h and t=72 h.
- 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). Test 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 nominal concentration of 3.2 mg/L to observe for any abnormal appearance of the algae compared to the control.

TEST CONCENTRATIONS
- Test concentrations in Combined Limit/Range-Finding Test: 0.099, 0.99, 9.9, 99 mg/L
- Results used to determine the conditions for the definitive study: yes, ErC50 is estimated to be between 9.9 and 99 mg/L (nominal concentrations).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Nov 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 5.2 - 5.9 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.19 - 0.25 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.21 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a TWA concentration of 0.51 mg/L when compared to the control.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
- Growth rate inhibition increased with increasing concentrations of Test Item at all test concentrations, resulting in 82% growth rate inhibition reduction at the highest test concentration. Statistically significant growth rate inhibition was found at all test concentrations. However, growth rate inhibition recorded at the lowest test concentration was below 10% and therefore considered to be biologically not relevant. The NOEC based on biological relevance was thus set at the TWA concentration of 0.21 mg/L.
- The measured concentrations in the reserve samples taken at the start of the test were 0.57, 2.1, 8.0, 27, and 89 mg/L at nominal concentrations of 0.99, 3.2, 9.9, 32 and 99 mg/L, respectively. At the end of the exposure period, the exposure concentrations decreased to 9.2 – 63% of initially measured concentrations.
- The concentrations measured in the solutions incubated with algae were comparable to those measured in their counterparts incubated without algae throughout the test. Therefore, it was concluded that the presence of the algae had no effect on the concentrations of the test item in test medium.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50 for growth rate inhibition (72h-ERC50) was 1.05 mg/L with a 95% confidence interval ranging from 1.04 to 1.06 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistical significance: Williams Multiple Sequential t-Test, 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 and the percentages of yield reduction versus the logarithms of the corresponding TWA concentrations of the test item.
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: Determination of Time Weighted Average Concentrations

Test item Nominal conc. (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
0.99	0.566	0.215	0.0896	0.21
3.2	2.07	0.434	0.193	0.51
9.9	7.98	4.43	0.732	3.2
9.9*	8.01*	4.45*	0.800*	3.2*
32	27.2	20.9	5.84	15
99	89.1	86.9	55.7	76

* incubated without algae.

Table 2: Growth Rate and Percentage Inhibition for the Total Test Period

Test item TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.783	0.0125	6
0.21	1.695	0.0366	3	4.9^#
0.51	1.413	0.0178	3	21*
3.2	1.059	0.0249	3	41*
15	0.571	0.0182	3	68*
76	0.325	0.0634	3	82*

* effect was statistically significant,^#effect was statistically significant however biologically not relevant (<10%).

Table 3: Growth Rate and Percentage Inhibition at Different Time Intervals

Test item TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.864		1.910		1.574
0.21	3	1.845	1.0	1.709	11	1.531	2.7
0.51	3	1.521	18	1.468	23	1.250	21
3.2	3	1.452	22	1.168	39	0.558	65
15	3	1.193	36	0.314	84	0.205	87
76	3	1.113	40	0.090	95	-0.228	114

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' section for details on validity criteria.

Conclusions:

The 72-h ErC50, ErC10 and NOErC for Pseudokirchneriella subcapitata was respectively 5.6 mg/L, 0.22 mg/L and 0.21 mg/L (based on biological relevance), based on Time Weighted Average exposure concentrations.

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 substance at nominal concentrations of 0.99, 3.2, 9.9, 32 and 99 mg/L (3 replicates per concentration) and an untreated control (6 replicates). Measured concentrations in the test solution decreased to 9.2 - 63% of initially measured at the end of the exposure period. Therefore, effect parameters were based on Time Weighted Average exposure concentrations. TWA concentrations were calculated to be 0.21, 0.51, 3.2, 15 and 76 mg/L. Growth rate inhibition increased with increasing test item concentration, resulting in 82% growth rate inhibition at the highest test concentration.

The EC50 for growth rate inhibition (72h-ErC50) was 5.6 mg/L , the 72h-ErC10 was 0.22 mg/L. The 72h-NOErC for growth rate inhibition was <0.21 and 0.21 mg/L based on statistical significance and biological relevance, respectively. The study met all validity criteria, and is considered reliable without restriction.