Methyl DL-serinate hydrochloride

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:

02 - 05 Mar 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Methyl DL-serinate hydrochloride

EC Number:

227-047-1

EC Name:

Methyl DL-serinate hydrochloride

Cas Number:

5619-04-5

Molecular formula:

C4H9NO3.ClH

IUPAC Name:

methyl 2-amino-3-hydroxypropanoate hydrochloride

Details on test material:

- Name of test material (as cited in study report): DL-Serinemethylester-HCl
- Substance type: white solid
- Physical state: solid
- Expiration date of the lot/batch: 11 June 2013
- Storage condition of test material: In original container, at room temperature (+10 to +30 °C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period).
The concentrations of the test item were analysed in the duplicate test media samples from all test concentrations at test start. At test end, the concentration of the test item was analysed only in the duplicate test media samples from the highest test concentration. From the control samples only one of the duplicate samples was analysed fiom each of both sampling times. The aged samples of the other treatment levels were not analysed, since they were below the NOEC determined in this test.

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 concentration of nominal 100 mg test item/L was prepared by dissolving 60 mg test item into 600 mL test water by intense stirring for 10 minutes. The pH of this test medium was adjusted from 6.8 to 8.0 with 1M NaOH. Appropriate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations. The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): cultivated in the laboratories of IBACON; original source: "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der 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

Test temperature:

23 - 24 °C

pH:

8 (start)
9.2 - 10.1 (end)

Nominal and measured concentrations:

control, 6.25, 12.5, 25, 50, and 100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Erlenmeyer flasks of 50 mL volume with 50 mL of test medium
- Initial cells density: 5 x 10^4 cells/mL
- Control end cells density: 131.611 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Blanks: One replicate of each test concentration and of the control was prepared without algae to provide a "blank" for the spectrophotometrical measurements. The additional replicates were incubated under the same conditions as described above. The blank values were subtracted from the absorption measured in the samples containing algae in order to eliminate absorption caused by test item.

GROWTH MEDIUM
- Standard medium used: yes, OECD medium


TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: temperature was measured daily, pH was measured at the start and the end of the study, light intensity was measured once during the test, the appearance of the test item in test water was determined daily in all test item concentrations.

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 6528 Lux (6250 - 6800 Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): suspensions were sampled after 24, 48 and 72 hours for determination of cell densities
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2x
- Range finding study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

other: EC50, EC10, NOEC, LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: EC50, EC10, NOEC, LOEC

Effect conc.:

> 97 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: EC50, EC10, NOEC, LOEC

Effect conc.:

> 22 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:

other: EC50, EC10, NOEC, LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

other: EC50, EC10, NOEC, LOEC

Effect conc.:

> 97 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

other: EC50, EC10, NOEC, LOEC

Effect conc.:

> 22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

The microscopic examination of the shape of the aIgaI cells after 72 hours of test duration did not show any difference between the aigae that had been growing at a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour E(r)C50 and the 72-hour E(y)C50, the corresponding EC10 values and where possible their 95%-confidence limits were calculated by Probit analysis. For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams Test (growth rate and yield),
respectively. The software used to perform the statistical analysis was Toxrat Professional, Version 2.09, Toxrat® Solutions GmbH, 2005

Any other information on results incl. tables

Analytical Results:

The quantification of the test item was performed using liquid chromatography (HPLC-method). In the freshly prepared test media test item concentrations were measured in the range from 81% to 106% of the nominal values. In the aged test media 5% of the nominal test concentrations were determined. Correct dosing could be demonstrated. However, since the test item is not stable under the conditions of the test, all biological endpoints were related to nominal, initial mean measured and geometric mean measured test concentrations.

Biological results:

The influence of DL-Serinmethylester-HCl on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose response test. The 72-hour NOE(r)C and the 72-hour NOE(y)C were determined to be at least 100 mg test item/L (nominal). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L, have not been tested according to the recommendations of the test guidelines. The 72-hour LOEC, the 72-hour EC10 and the 72 -hour EC50 for the parameters growth rate and yield were clearly higher than the highest test concentration of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of DL-Serinmethylester-HCl up to the highest test concentration of nominal 100 mg test item/L. Even if instability of the test item was observed during the algae study, the integrity of the study is not influenced since degradation of the test item would also occur in the environment.

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

Nominal concentration [mg test item/L]	Flask No.	Density of algal cells/mL [10^4] after
		24 hours	48 hours	72 hours
Control	1	5.148	38.409	138.194
	2	5.148	35.551	140.013
	3	4.758	38.669	125.46 1
	4	5.278	36.200	129.619
	5	4.888	36.980	128.3 19
	6	5.018	35.161	128.059
	m	5.039	36.828	131.611
	S	0.191	1.464	5.986
6.25	1	4.888	40.748	134.686
	2	4.888	3 8.539	133.646
	3	5.148	40.488	130.918
	m	4.974	39.925	133.083
	S	0.150	1.207	1.946
12.5	1	4.368	37.110	129.748
	2	4.628	40.748	135.205
	3	4.238	42.957	144.430
	m	4.411	40.272	136.461
	S	0.198	2.952	7.421
25	1	4.368	39.838	144.690
	2	4.498	42.047	139.883
	3	3.978	42.957	149.757
	m	4.282	41.614	144.777
	S	0.270	1.604	4.938
50	1	4.238	40.878	144.170
	2	4.108	37.630	155.604
	3	4.628	42.307	158.333
	m	4.325	40.272	152.702
	S	0.270	2.397	7.514
100	1	4.238	41.268	157.163
	2	4.23 8	39.708	155.864
	3	4.368	38.149	147.938
	m	4.282	39.708	153.655
	S	0.075	1.559	4.993

m = mean, s = standard deviation (at test start 5000 algal cells/mL were incubated)

Table 2: Growth Rates µ and Percentage Inhibition of µ During the Test Period

Nominal concentration [mg test item/L]	Growth rates µ [1/day] and % inhibition of µ
	0 - 24 hours			0 - 48 hours		0 - 72 hours
	µ	%		µ	%	µ	%
Control	2.3 10	0.0		2.149	0.0	1.857	0.0
6.25	2.297	0.5		2.190	-1.9	1.861	-0.2
12.5	2.177	5.8	*	2.193	-2.1	1.869	-0.6
25	2.146	7.1	*	2.211	-2.8	1.889	-1.7
50	2.156	6.6	*	2.194	-2.1	1.907	-2.7
1 O0	2.147	7.0	*	2.187	-1.8	1.909	-2.8

negative values in "% inhibition" indicate an increase in growth relative to that of the control;

* mean value significantly different from the control (tested with Williams Test, alpha = 0.05, one-sided)

Table 3: Yield y and Percentage Inhibition of y During the Test Period

Nominal concentration [mg test item/L]	Yield y [x * 10^4 cells/mL] and % inhibition of y
	0 - 24 hours			0 - 48 hours		0 - 72 hours
	y	%		y	%	y	%
Control	4.539	0.0		36.328	0.0	131.111	0.0
6.25	4,474	1.4		39.425	-8.5	132.583	-1.1
12.5	3.91 1	13.8	*	39.772	-9.5	135.961	-3.7
25	3.782	16.7	*	41.114	-13.2	144.277	-10.0
50	3.825	15.7	*	39.772	-9.5	152,202	-16.1
1 O0	3.782	16.7	*	39.208	-7.9	153.155	-16.8

negative values in "% inhibition" indicate an increase in growth relative to that of the control;

* mean value significantly different from the control (tested with Williams Test, alpha = 0.05, one-sided)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study no adverse effects to Pseudokirchneriella subcapitata were observed. Analytical measurement showed a strong decrease of the test item concentrations during the 72 hours test period. Thus, results were based on nominal, initial measured, and geometric mean measured concentrations. The EC50 and NOEC were determined to be >100 mg/L and = 100 mg/L, respectively. All validity criteria were fulfilled.

Executive summary:

The influence of the test item DL-Serinemethylester on the growth of the green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline No. 201, 2006.

The nominal test concentrations were 6.25, 12.5, 25, 50, 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 5% of the nominal values. Even if instability of the test item was observed during the algae study, the integrity of the study is not influenced, since degradation of the test item would also occur in the environment.

Since the test item is not stable under the conditions of the test, all biological endpoints were related to nominal, initial mean measured and geometric mean measured test concentrations.

The 72-hour NOE(r)C and the 72-hour NOE(y)C were determined to be at least 100 mg test item/L (nominal). The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L, have not been tested according to the recommendations of the test guidelines. The 72-huur LOEC, the 72-hour EC10 and the 72-hour EC50 for the parameters growth rate and yield were clearly higher than the highest test concentration of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of DL-Serinemethylester-HCI up to the highest test concentration of nominal 100 mg test item/L.

The EC values were calculated for both parameters, the algal growth rate (µ) and yield (y), after 72 hours test duration:

Parameter (0-72 h)	Growth rate µ (mg/L)	Yield y (mg/L)
EC50 (nominal/initial measured/geometric mean measured)	>100 / >97 / >22	>100 / >97 / > 22
EC10 (nominal/initial measured/geometric mean measured)	>100 / >97 / >22	>100 / >97 / >22
NOEC (nominal/initial measured/geometric mean measured)	>100 / >97 / >22	>100 / >97 / >22
LOEC (nominal/initial measured/geometric mean measured)	>100 / >97 / >22	>100 / >97 / >22