DL-serinohydrazide monohydrochloride

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:

19 Jan - 02 Feb 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Federal Office of Public Health, Bern, Switzerland

Test material

Test material information

Constituent 1

Reference substance name:

DL-serinohydrazide monohydrochloride

EC Number:

259-839-8

EC Name:

DL-serinohydrazide monohydrochloride

Cas Number:

55819-71-1

Molecular formula:

C3H9N3O2.ClH

IUPAC Name:

2-amino-3-hydroxy-propanehydrazide;hydrochloride

Details on test material:

- Name of test material (as cited in study report): DL-Serine HCl
- Substance type: white solid
- Physical state: solid
- Expiration date of the lot/batch: 06 Dec 2001
- Storage condition of test material: in refrigerator at about 4 °C, under N2

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For the analysis of the actual test item concentrations, duplicate samples without algae were taken from each test concentration and the control just before test start and after 72 hours (end of the test). For the 72-hour stability samples additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as in the actual test but without algae.
All the samples were directly analyzed after sampling since a significant loss of test item was determined in deep-frozen samples (results of a pre-experiment, without GLP).
The concentrations of the test item DL-Serine hydrazide HCl were analyzed in the duplicate test media samples from the test concentrations of nominal 1.0 to 100 mg/L from both sampling times (0 and 72 hours). From the control samples and the samples of the lowest test concentration of nominal 0.32 mg/L, only one of the duplicate samples was analyzed from the corresponding sampling times.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest test item concentration of nominal 100 mg/L was prepared by dissolving 80 mg of the test item completely in 800 mL test water by intense stirring for 5 minutes at room temperature. Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media of the lower test item concentrations. The test media were prepared just before addition of algae (= start of the test). The actual concentration of the test item in the test media was analytically determined.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: No. 86.81 SAG
- Source (laboratory, culture collection): cultivated in the laboratories of RCC; original source: "Sammlung von Algenkulturen" (Experimentelle Phykologie und Sammlung von algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universät Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): 3 days

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/L) as CaCO3

Test temperature:

22 °C

pH:

7.0 - 7.9 (start)
7.1 - 8.2 (end)

Nominal and measured concentrations:

control, 0.32, 1.0, 3.2, 10, 32, and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks filled with 15 mL suspension
- Initial cells density: 10^4 cells/mL
- Control end cells density: 117 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic test water according to test guidelines
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was recorded daily. In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 3.2 mg/L). The shape of the algal cells was microscopically examined.

OTHER TEST CONDITIONS
- Photoperiod: continuously illumination
- Light intensity and quality: 8000 Lux (7600 - 8400 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): algal cell densities were determined at 24, 48, and 72 hours
- Determination of cell concentrations: electronic particle counter

TEST CONCENTRATIONS
- Range finding study: yes

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 5.7 - 14

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

47 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 31 - 84

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.7 - 3.0

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 2.4 - 8.7

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The microscopic examination of the aigal cells after 72 hours test period showed no difference between the algae growing in the test concentration of nominal 3.2 mg/l and the algal cells in the control. The shape and size of the algal cells growing in test media containing the test item at up to this test concentration were obviously not affected.

Any other information on results incl. tables

Analytical results:

The analytical measurements demonstrated the correct dosage of the test item. During the test period of 72 hours, a strong decrease of the test item concentrations in the test media was determined. At the end of the test, the test item concentrations were 0.9% and 48% of the nominal values at the test concentrations of nominal 32 mg/L and 100 mg/L, respectively. At the test concentrations of nominal 0.32 to 10 mg/L, the concentrations of the test item were at the end of the test below the limit of quantification of 0.25 mg/L. The losses were obviously caused by a catalytic degradation of the test item in presence of the trace elements in the test water since no degradation of the test item was observed in the test water used for the acute Daphnia toxicity test with the test item over a period of 48 hours (RCC Study No. 793866) and in a 96 hour stability test (pre-experiment without GLP). The fast catalytic degradation of the test item DL-Serine hydrazide HCl will occur also under natural conditions where trace elements are always present (otherwise no algal growth would occur). Due to the fast degradation of the test item with half-life periods below one day at the lower test concentrations, the parent or the degradation products might have been responsible for the toxic effect to Scenedesmus subspicatus. The biological results are therefore related to the nominal test concentrations.

Biological results:

The test item had a statistically significant inhibitory effect on the growth (i.e. biomass) of Scenedesmus subspicatus after the exposure period of 72 hours at nominal concentrations of 3.2 mg/L and above (results of a Dunnett- test, one-sided, alpha = 0.05). Thus, this test concentration was determined as the 72-hour LOEC (lowest concentration tested with toxic effects). The growth rate µ was statistically significantly reduced first at the next higher test concentration of 10 mg/L. The 72-hour NOEC (highest concentration tested without toxic effects after a test period of 72 hours) was determined to be 1.0 mg/L, since up to and including this test concentration, the mean biomass and growth rate of the algae was not statistically significantly lower than in the control.

Table 1: Algal cell densities during the test period of 72 hours

Nominal concentration (mg/L)	Flask No.	Density of algal cells (cell number x 10,000/mL) after
		24 h		48 h		72 h
control	1	7.4	7.3	26.3	26.1	116.1	112.2
	2	7.5	8.0	27.6	27.1	115.6	114.3
	3	6.8	6.3	25.7	25.6	118.0	112.6
	4	7.1	6.8	25.2	24.9	122.4	117.6
	5	6.6	6.3	23.7	23.2	118.9	115.7
	6	5.8	6.2	23.9	24.5	120.5	120.4
	m	6.84		25.32		117.03
	s	0.64		1.40		2.69
	n	6		6		6
0.32	1	6.2	6.1	26.6	26.3	126.9	128.7
	2	6.9	6.3	28.3	28.0	114.0	128.0
	3	6.3	5.5	28.5	27.8	127.5	138.9
	m	6.22		27.58		127.33
	s	0.35		0.98		6.11
	n	3		3		3
1.0	1	5.4	5.2	24.4	24.5	109.9	113.0
	2	5.8	5.4	25.4	24.2	108.2	112.3
	3	6.6	5.7	27.8	27.5	115.4	116.6
	m	5.68		25.63		112.57
	s	0.43		1.76		3.03
	n	3		3		3
3.2	1	4.3	4.5	20.0	20.4	98.1	96.1
	2	4.6	4.2	19.9	19.8	111.9	108.2
	3	3.8	3.7	17.9	18.8	100.2	97.6
	m	4.18		19.47		102.02
	s	0.38		0.98		7.02
	n	3		3		3
10	1	3.3	3.8	12.0	11.8	37.5	32.4
	2	3.6	3.1	14.7	14.4	54.3	53.4
	3	3.5	3.0	12.8	12.7	37.8	34.7
	m	3.38		13.07		41.68
	s	0.15		1.35		10.56
	n	3		3		3
32	1	3.2	2.9	2.7	2.7	24.9	21.2
	2	3.1	2.9	2.4	2.7	11.0	7.6
	3	2.9	2.9	2.3	2.4	9.8	7.1
	m	2.98		2.53		13.60
	s	0.08		0.18		8.19
	n	3		3		3
100	1	2.8	3.0	3.0	2.7	8.3	7.2
	2	3.2	2.8	2.7	2.6	9.1	5.6
	3	2.7	2.8	2.8	2.3	4.9	4.2
	m	2.88		2.68		6.55
	s	0.13		0.15		1.74
	n	3		3		3

m = mean value; s = standard deviation; n = number of flasks; at the start 10,000 algal cells/mL were incubated

Table 2: Areas under the growth curves (AUC) and percentage inhibition of AUC (I(AUC)) during the test period

Nominal concentration (mg/L)	Areas under the Growth Curves (AUC) and % inhibition of AUC
	0-24 h		0-48 h		0-72 h
	AUC	I(AUC) (%)	AUC	I(AUC) (%)	AUC	I(AUC) (%)
control	70	0.0	432	0.0	2116	0.0
0.32	63	10.7	444	-2.8	2279	-7.7
1.0	56	19.8	408	5.6	2042	3.5
3.2	38	45.5	298	31.0	1732	18.2
10	29	59.2	202	53.2	835	60.5
32	24	66.0	66	84.7	236	88.9
100	23	67.8	65	84.9	152	92.8

Table 3: Growth rates (µ) and percentage inhibition of µ (I(µ)) during the test period

Nominal concentration (mg/L)	Growth Rate µ (1/day) and % inhibition of µ
	0-24 h		0-48 h		0-72 h
	µ	I(µ) (%)	µ	I(µ) (%)	µ	I(µ) (%)
control	1.92	0.0	1.62	0.0	1.59	0.0
0.32	1.83	4.9	1.66	-2.7	1.62	-1.8
1.0	1.74	9.6	1.62	-0.4	1.57	0.8
3.2	1.43	25.6	1.48	8.1	1.54	2.9
10	1.22	36.5	1.28	20.5	1.24	22.1
32	1.09	43.1	0.46	71.3	0.83	47.5
100	1.06	44.9	0.49	69.5	0.62	61.1

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study inhibitory effects to the green alga Scenedesmus subspicatus were observed after 72 hours. Analytical measurement showed a strong decrease of the test item concentrations during the 72 hours test period. The losses were obviously caused by a catalytic degradation of the test item in presence of the trace elements in the test water. Thus, results were based on nominal concentrations and the EC50, LOEC and NOEC determined to be 8.8, 3.2, and 1.0 mg/L for biomass and 47, 10, and 3.2 mg/L for growth rate, respectively. All validity criteria were fulfilled.

Executive summary:

The influence of the test item DL-Serine hydrazide HCI on the growth of the green algal species Scenedesmus subspicatus was investigated in a 72-hour static test according to the EU Commission Directive 92/69/EEC, C.3, 1992, and the OECD Guideline No. 201, 1984.

The nominal test concentrations were 0.32, 1.0, 3.2, 10, 32, and 100 mg/L. Additionally, a control was tested in parallel.

The analytical measurements demonstrated the correct dosage of the test item. During the test period of 72 hours, a decrease of the test item concentrations in the test media was determined. At the end of the test, the test item concentrations were 0.9% and 48% of the nominal values at the test concentration of nominal 32 mg/L and 100 mg/L, respectively. At the test concentrations of nominal 0.32 to 10 mg/L, the concentrations of the test item were at the end of the test below the limit of quantification of 0.25 mg/L. The losses could be due to a catalytic degradation of the test item in presence of the trace elements in the test water, since no degradation of the test item was observed in the test water used for an acute Daphnia toxicity test with the test item over a period of 48 hours.

The fast catalytic degradation of the test item DL-Serine hydrazide HCI will occur also under natural conditions where trace elements are always present (otherwise no algal growth would occur). Due to the fast degradation of the test item with half-life periods below one day at the lower test concentrations, the parent or the degradation products might have been responsible for the toxic effect to Scenedesmus subspicatus. The biological results are therefore related to the nominal test concentrations.

The test item DL-Serine hydrazide HCI had a statistically significant inhibitory effect on the growth (i.e. biomass) of Scenedesmus subspicatus after the exposure period of 72 hours at concentrations of nominal 3.2 mg/L (= 72-hour LOEC: lowest concentration tested with toxic effects) and above. The 72-hour NOEC (highest concentration tested without toxic effects) was determined to be nominal 1.0 mg/L, since up to this test concentration the mean biomass and growth rate of the algae were statistically not significantly lower than in the control.

The EC values were calculated for both parameters, the algal biomass (b) and the growth rate (p), after 72 hours test duration:

Parameter (0-72 h)	Biomass b (mg/L)	Growth rate p (mg/L)
EC50 (95%-confidence limits)	8.8 (5.7-14)	47 (31-84)
EC10 (95%-confidence limits)	1.7 (0.7-3.0)	5.3 (2.4-8.7)
EC90 (95%-confidence limits)	45 (26-114)	>100
NOEC	1.0	3.2
LOEC	3.2	10

 

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