Serine, 2-[(2,3,4-trihydroxyphenyl)methylene]hydrazide, hydrochloride (1:1)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Remarks:

Conducted according to guideline in effect at the time of study conduct

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: U.S. Environmental Protection Agency Series 850 – Ecological Effects Test Guidelines OCSPP Guideline 850.4500 (2012)

Deviations:

no

Remarks:

Conducted according to guideline in effect at the time of study conduct

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-3-hydroxy-N'-[(E)-(2,3,4-trihydroxyphenyl)methylidene]propanehydrazide hydrochloride

EC Number:

940-411-0

Cas Number:

1353749-74-2

Molecular formula:

C10H14ClN3O5

IUPAC Name:

2-amino-3-hydroxy-N'-[(E)-(2,3,4-trihydroxyphenyl)methylidene]propanehydrazide hydrochloride

Details on test material:

- Purity: 100%

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the active substance. At test initiation, samples were collected from each test concentration and control solution from the middle layer of solution prior to distribution into test chambers. At exposure termination, the biological replicates from each respective test concentration and blank control solution were pooled and then sampled. The abiotic control replicate was also sampled. Two sets of samples were collected at each interval. For each sample, a 9 mL aliquot of the sample was transferred to a vial containing 1 mL of acidified methanol. Algae were removed from the 72 hour samples by centrifugation prior to analysis. One set was processed immediately for analysis. The second set was stored refrigerated for possible future analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
A primary stock solution was prepared by dissolving 0.0100 g of the test substance in freshwater AAP medium to a total volume of 1000 mL, for a nominal concentration of 10 milligrams per litre. The stock was mixed by sonicating for 20 minutes and inverting to mix, and appeared clear and yellow in colour after mixing. Additional test solutions were prepared in freshwater AAP medium at nominal concentrations of 0.041, 0.12, 0.37, 1.1 and 3.3 mg/L by proportionally diluting the primary stock solution, which was used as the test solution for the highest treatment level. The test solutions were inverted to mix. The 0.041, 0.12, and 0.37 mg/L solutions appeared clear and colourless and otherwise unremarkable at the time of preparation. The remaining test solutions appeared clear and yellow in colour. At test termination, no surface slicks or particulates were noted in any test solution.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: freshwater green alga, Pseudokirchneriella subcapitata. Original algal cultures were obtained from the University of Toronto Culture Collection and had been maintained in culture medium at Wildlife International, Easton, Maryland.
- Age of inoculum (at test initiation): Algal cells used in this test were obtained from Wildlife International cultures that had been actively growing in culture medium for at least two weeks prior to test initiation.

ACCLIMATION PERIOD: Algal cells for this study were taken from a culture that had been transferred to fresh medium three days prior to test initiation.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23.5 to 25.3°C

pH:

The pH of test solutions at test initiation ranged from 7.0 to 7.2, and at exposure termination the pH of the biotic test solutions ranged from 7.1 to 7.9.

Salinity:

freshwater

Nominal and measured concentrations:

Nominal Concentrations: 0.041, 0.12, 0.37, 1.1, 3.3 and 10 mg/L
Geometric Mean, Measured Concentrations: 0.0012, 0.036, 0.20, 0.32, 0.85 and 4.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flask containing 100 mL of test solution and plugged with foam stoppers
- Initial cells density: nominal concentration of approximately 10000 cells/mL at test initiation.
- Control end cells density: 1836253 cells/mL
- No. of organisms per vessel: initial cell count - 1.009 e6/vessel
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes (Freshwater (AAP) Medium)

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: continuous
- Light intensity and quality: cool-white fluorescent lighting at a target intensity of 4300 ± 10% lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Samples were collected at approximately 24-hour intervals during the 72-hour exposure for examination
- Determination of cell concentrations: electronic particle counter
- Morphology: cells were examined microscopically for atypical cell morphology

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

0.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.62 to 1.29 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

0.23 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.14 to 0.37 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): At exposure termination, cells in the blank control and the 0.0012, 0.036, 0.20, 0.32 and 0.85 mg/L treatment groups were observed to be normal in size, shape, and colour. Cells in the 4.6 mg/L treatment group appeared enlarged compared to the control cells. There was no evidence of aggregation or adherence to the test chambers in any test group.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Reported statistics and error estimates:

The calculation of areas under the growth curve, growth rates, yields and percent inhibition values, as well as all statistical analyses, were conducted using SAS Version 8.2. The 72-hour area under the growth curve, growth rate and yield data were evaluated for normality and homogeneity of variance (α = 0.01) using the Shapiro-Wilk’s and Levene’s tests, respectively. Where the data did not meet the assumptions for normality or homogeneity of variance, log transformations (natural log) were performed to correct any condition of non-normality or unequal error variances. The treatment groups were compared to the control using ANOVA and Dunnett’s test (α = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC and LOEC relative to each parameter at 72 hours.

Any other information on results incl. tables

Cell density increased in the blank control by a factor of 185 in 72 hours. The coefficient of variation of average specific growth rates during the whole exposure period (0-72 hour) in replicates of the blank control was 3.2%. In addition, the mean percent coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) was 7.9%. Therefore, the test was considered to be valid.

To determine if the test substance had an algistatic or algicidal effect, recovery was initiated for the geometric mean, measured concentrations of 0.32, 0.85 and 4.6 mg/L. In the recovery phase it was demonstrated that the effects of the test substance were algistatic (reversible) rather than algicidal, to P. subcapitata at concentrations less than or equal to 4.6 mg/L based on greater than 16X increase of healthy cells within seven days.

Geometric Mean, Measured Concentration (mg/L)	Mean 0-72 hour growth rate (hours-1)	% Inhibition^a
Blank control	0.0723	--
0.0012	0.0755	-4
0.036	0.0721	0
0.20	0.0688	5
0.32	0.0538*	26
0.85	0.0274*	62
4.6	0.0190*	74
Geometric Mean, Measured Concentration (mg/L)	Mean 0-72 hour area under growth curve	% Inhibition^a
Blank control	31,290,387	--
0.0012	40,470,600	-29
0.036	32,031,468	-2
0.20	26,649,489	15
0.32	10,941,195*	65
0.85	1,711,404*	95
4.6	999,210*	97
a  Relative to the blank control. * Treatment group mean is significantly different from blank control (Dunnett’s test,p< 0.05).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
72-hour EbC50 = 0.23 mg/L
72-hour ErC50 = 0.90 mg/L
72-hour NOEC = 0.20 mg/L
72-hour LOEC = 0.32 mg/L

Executive summary:

Pseudokirchneriella subcapitata was exposed to six concentrations of the test substance and evaluated for effects on area under the growth curve, growth rate and yield according to OECD Guideline 201. The E_bC₅₀ and E_rC₅₀ were calculated to be 0.23 and 0.90 mg/L, respectively. Dunnett’s test indicated that mean area under the growth curve, growth rate and yield were significantly reduced (p<0.05) relative to the blank control at the 0.32, 0.85 and 4.6 mg/L test levels. Consequently, for all endpoints, the NOEC and LOEC were determined to be 0.20 and 0.32 mg/L, respectively. The test substance was determined to be algistatic at concentrations less than or equal to 4.6 mg/L, the highest concentration tested.