Glycine hydrochloride

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on March 23, 2006; Annex 5 corrected: July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

7 December 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Two replicates of each sample were deep-frozen (< -20°C) directly after preparation and after 24h, 48h and 72h exposure, respectively, and stored in a freezer until analysis. Before freezing the samples after 24h, 48h and 72h exposure, the algae were separated from the medium by centrifugation at a low g-force (4000g for 20 minutes), sufficient to settle the algae. Additionally, analysis of the item concentration not inoculated with algae was performed. One replicate was given to the analytical laboratory for analysis. For analytical measurement the samples were thawed at RT. The second replicate was used as backup.

Vehicle:

no

Details on test solutions:

A preparation of the test medium with a test item concentration of 100 mg/L was freshly prepared. For that purpose, the test item was weighed in a calibrated flask and vehicle was added. The preparation was stirred with a magnetic stirrer for 1 hour. The pH was adjusted to approx. 7.9 with NaOH(1 M).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source: ,,Pflanzenphysiologisches Institut der Universitat Göttingen", Germany
- Age: three days old pre-culture

ACCLIMATION
- Acclimation period: no, same as test condition
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3

Test temperature:

22.6 to 23.7°C

pH:

7.88 to 8.39

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentration: 91.5 - 99 mg/L, details in Table 2 and 3

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: open
- Material, fill volume: glass, 100 mL
- Aeration: no
- Initial cells density: 10,000 cells/mL
- Control end cells density: 708,843 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
Reconstituted water was used as vehicle. The following components (analytical grade) were dissolved in fully demineralized water (Milli-Q" Water Purification System, Merck Millipore, Darmstadt, Germany) to obtain the following concentrations:
Nutrient [mg/L]
NH4CI 15.0
MgCb * 6H2O 12.0
CaCh * 2H2O 18.0
MgSCM * 7H2O 15.0
KH2PO4 1.60
FeCb * 6H2O 0.0640
Na2EDTA * 2H2O 0.100
H3BO3 0.185
MnCl2*4H2O 0.415
ZnCb 0.00300
CoCI2 * 6H2O 0.00150
CuCb * 2H2O 0.00001
Na2MoO4 * 2H2O 0.00700
NaHCOs 50.0
The vehicle was aerated for at least 24 hours prior preparation of the test item concentrations.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, with NaOH (1 M)
- Photoperiod: continuously
- Light intensity and quality: 7274 - 7371 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter (Coulter-Counter Z2, Beckman Coulter GmbH, Krefeld, Germany)
- Other: pH and light intensity measured at start and end of test, temperature measured continuously

TEST CONCENTRATIONS
Range finding study
- Test concentrations: 100 mg/L
- Results used to determine the conditions for the definitive study: yes, no effects of growth inhibition observed.

Reference substance (positive control):

yes

Remarks:

(potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and Yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate and Yield

Details on results:

In the control group, the biomass increased by a factor of 70.9 over 72 hours. Therefore, the validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control group (section-by-section growth rates) during 72 hours was 28.8%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control group after 72 hours was 2.8%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.
Due to the fact, that all test item concentrations (inoculated with and without algae) were >80% of the nominal concentration throughout the study, the EC values were based on the nominal concentrations.

Results with reference substance (positive control):

The test item Art. 104864 (Potassium dichromate) showed 72h EC50 of 1.10 mg/L for growth rate and 0.48 mg/L for yield. The EC50 for growth rate and yield obtained in this study are in the range of ± two standard deviation of the current historical means.
Therefore, this study confirms the sensitivity and reliability of the test system used in the test facility.

Reported statistics and error estimates:

The calculated growth rates and yield were tested for significant differences compared to the control values by Student-t test. The software used to perform statistical analysis was ToxRat (ToxRat Professional Version 2.10, ToxRat Solutions GmbH, Alsdorf, Germany).

Table 1: Algal biomass (cell number) after 24, 48 and 72 hour exposure:

Nominal concentration [mg/L]	Biomass of algae (Cell number per mL*)
Control		0 hours	24 hours	48 hours	72 hours
		1.0000	5.2650	26.3196	70.1280
		1.0000	4.7637	24.3540	61.1397
		1.0000	5.1300	31.4433	87.2757
		1.0000	4.7493	28.3653	70.5177
		1.0000	4.6593	26.1630	66.6720
		1.0000	4.8690	26.5986	69.5727
	Mean	1.0000	4.9061	27.2073	70.8843
	SD	0.00000	0.23920	2.43619	8.76134
	CV	0.0	4.9	9.0	12.4
100.0		0 hours	24 hours	48 hours	72 hours
		1.0000	4.6467	29.5677	127.9593
		1.0000	4.4667	31.0923	135.3303
		1.0000	4.5324	32.5440	134.3673
		1.0000	4.5900	32.8743	129.8403
		1.0000	4.4604	32.5917	128.2293
		1.0000	4.4064	30.9987	127.0773
	Mean	1.0000	4.5171	31.6115	130.4673
	SD	0.00000	0.08984	1.28431	3.52258
	CV	0.0	2.0	4.1	2.7

*Cell number divided by factor 10000

 

Table 2: Nominal and corresponding analytical concentrations of samples inoculated with algae

Nominal test item concentration [mg/L]	Analytical results
	0h^1		24h		48h		72h
	Meas. Cone. [mg/L]	[%]^2	Meas. Cone. [mg/L]	[%]^2	Meas. Cone. [mg/L]	[%]^2	Meas. Cone. [mg/L]	[%]^2
0.0	<LOD	-	<LOD	-	<LOD	-	<LOD	-
100.0	96.215	96.2	95.710	95.7	94.280	94.3	91.451	91.5

¹Measured without algae

²Rounded to the first decimal place

 

 

Table 3: Nominal and corresponding analytical concentrations of samples inoculated without algae

Nominal test item concentration [mg/L]	Analytical results
	Oh		24h		48h			72h
	Meas. Cone. [mg/L]	[%]^1	Meas. Cone. [mg/L]	[%]^1	Meas. Cone. [mg/L]	[%]^1	Meas. Cone. [mg/L]		[%]^1
100.0	96.215	96.2	97.377	97.4	95.955	96.0		98.996	99.0

¹Rounded to the first decimal place

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study according to OECD 201, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50 values for growth rate and yield are >100 mg/L and, thus, could not be determined in this study. The NOEC value for growth rate and yield is 100 mg/L.

Executive summary:

The objective of the present study according to OECD 201 was to determine the effect of the test item on the growth and growth rate of the green algae species Pseudokirchneriella subcapitata. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The initial algae cell density in each replicate was 10,000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. Samples of the test and control media were taken directly after preparation and then daily and stored at < -20°C until further analysis of the test item concentration via HPLC with UV detection. The recoveries of the test item concentrations in all test medium samples with algae ranged from 91.5% to 96.2%. The recoveries of the test item concentrations in all test media without algae ranged from 96.0% to 99.0% of the nominal concentration over the time period of 72h and, thus, the test item was stable in the test medium during the study. Due to the fact, that all test item concentrations (inoculated with and without algae) were >80% of the nominal concentration throughout the study, the EC values were based on the nominal concentrations. No significant effect on the inhibition of growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L. For the test item the following EC values were determined: The 72h EC50 values for growth rate and yield are >100 mg/L and, thus, could not be determined in this study. The NOEC value for growth rate and yield is 100 mg/L.

Description of key information

Under the conditions of the study according to OECD 201, an aqueous solution of nominal 100 mg/L of the test item revealed no toxicity to algae. The 72h EC50 values for growth rate and yield are >100 mg/L and, thus, could not be determined in this study. The NOEC value for growth rate and yield is 100 mg/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

The objective of the present study according to OECD 201 was to determine the effect of the test item on the growth and growth rate of the green algae species Pseudokirchneriella subcapitata. The study comprised of one control group and one test item group with six replicates each. A nominal test item concentration of 100 mg/L was prepared in reconstituted water. The initial algae cell density in each replicate was 10,000 cells per mL. The growth of the algae was determined over an exposure period of 72 hours and compared with the growth of the control group. Samples of the test and control media were taken directly after preparation and then daily and stored at < -20°C until further analysis of the test item concentration via HPLC with UV detection. The recoveries of the test item concentrations in all test medium samples with algae ranged from 91.5% to 96.2%. The recoveries of the test item concentrations in all test media without algae ranged from 96.0% to 99.0% of the nominal concentration over the time period of 72h and, thus, the test item was stable in the test medium during the study. Due to the fact, that all test item concentrations (inoculated with and without algae) were >80% of the nominal concentration throughout the study, the EC values were based on the nominal concentrations. No significant effect on the inhibition of growth rate and yield of the algae was observed at a nominal concentration of 100 mg/L. For the test item the following EC values were determined: The 72h EC50 values for growth rate and yield are >100 mg/L and, thus, could not be determined in this study. The NOEC value for growth rate and yield is 100 mg/L (reference 6.1.5-1).