Sodium hydrogen substituted amino acid

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:

30 July 2018 - 21 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

A correction factor of 2.52 was used to correct for the composition of the test item, i.e. the water content. Concentrations are expressed as “mg a.i./L” when referring to the water-free fraction of the test item.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: from all test concentrations and the control
- Sampling method: 1.95 mL samples were taken at t=0 h, t=24 h and t=72 h from the approximate centre of the test vessels.
- Sample storage conditions before analysis: in a freezer (≤-15°C)

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: preparation of test solutions started with the highest concentration of 100 mg a.i./L applying a 15- to 16-minute period of magnetic stirring to accelerate the dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: test medium without test item or other additives.
- Evidence of undissolved material: no, all test solutions were clear and colorless at the end of the
preparation procedure.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium 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. 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium 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.

ACCLIMATION: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

22-23°C

pH:

At the start of the test: 6.7-8.1
At the end of the test: 7.6-8.1

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 mg a.i./L
Measured: concentrations were 108-112%, 109-114% and 108-113% of nominal concentrations at t=0, t=24 and t=72 respectively. Measured concentrations at t=24 h and t=72 h were between 96 and 104% of measured concentrations at t=0 showing that the test item concentrations remained stable during the exposure period. Based on these results, effect parameters were based on nominal test item concentrations. For details on analytical results see table 1 in 'any other information on materials and methods incl. tables'

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: 216.5 x 10^4 cells/mL
- Replicates:
3 replicates of each test concentration;
6 replicates of the control;
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-medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water was used to formulate the medium
- Culture medium different from test medium: yes, M1-medium was used as stock culture medium
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test, temperature of the medium was measured continuously in a temperature control vessel

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 85 to 86 μE.m^-2.s^-1.
- Other: 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:
- 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.
- Other: Appearance of the cells was checked at the end of the final test; microscopic observations were performed on the highest test concentration and the control to observe for any abnormal appearance of the algae.

RANGE-FINDING STUDY:
- Test concentrations: three replicates were exposed to nominal concentrations of 0.10, 1.0, 10 and 100 mg a.i./L and a control
- Results used to determine the conditions for the definitive study: yes, a dose-related inhibition of algal growth rate was found at the two highest test concentrations at the end of the test. At the highest test concentration this resulted in 9.1% inhibition. Based on these results, the test concentrations for the definitive study were determined.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (July 2018)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: based on biological relevance

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

53 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95%-CI: 45-62 mg/L

Details on results:

- Any abnormal observations: no, microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.
- Inhibition of growth rates increased with increasing concentration of the test item from 10 mg a.i./L upwards resulting in 15% inhibition at 100 mg a.i./L. Statistically significant inhibition of growth rates was found at all test concentrations. However, growth rate inhibition was considered to be biologically not relevant at 32 mg a.i./L and below, where the observed inhibition was below 10%. The NOEC based on biological relevance was thus set at 32 mg a.i./L.

- The experimental conditions during the study (pH and temperature) were within the limits prescribed by the study plan.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Test concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and a control
- 72h-ErC50: 0.90 mg/L (95% confidence interval ranging from 0.88 to 0.93 mg/L).
- Other: results were within the historical range of the test facility (0.82 - 2.3 mg/L) thereby showing that the batch of algae was adequate for testing.

Reported statistics and error estimates:

For determination of the NOEC and the ECx, an effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsht-
test After Bonferroni-Holm, 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 versus the logarithms of the corresponding nominal concentrations of the test item.
The EC20- and EC50-values for growth rate inhibition could not be determined because the observed effects were below 20%.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 2 Growth rate and percentage inhibition for the total test period

Sodium hydrogen substituted amino acid solution Nominal conc. (mg a.i./L)	Mean	Std. Dev.	n	%Inhibition
Control	1.792	0.0196	6
10	1.729	0.0113	3	3.5^#
18	1.715	0.0152	3	4.3^#
32	1.659	0.0077	3	7.4^#
56	1.606	0.0225	3	10 *
100	1.532	0.0584	3	15 *

* Effect was statistically significant.;

^#Effect was statistically significant but biologically not relevant (<10%).

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks'

Conclusions:

Based on the results of a short-term aquatic toxicity study, performed according to OECD 201 and GLP principles, Sodium hydrogen substituted amino acid solution inhibited growth of Pseudokirchneriella subcapitata at analytically confirmed nominal concentrations of 10 mg a.i./L and higher. The 72 h-ErC50, ErC10 and NOEC were determined to be >100 mg a.i./L, 53 mg a.i./L and 32 mg a.i./L (based on biological significance), respectively.

Executive summary:

The objective of the study was to evaluate Sodium hydrogen substituted amino acid solution for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀and EC₅₀ for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of Sodium hydrogen substituted amino acid solution tested was a clear colourless solution with a purity of 35.8% and completely soluble in test medium at the concentrations tested. A correction factor of 2.52 was used to correct for the water content of the test item. Concentrations are expressed as “mg a.i./L” when referring to the water-free fraction of the test item.

A final test was performed based on the results of a preceding range-finding test.Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg a.i./L. The initial algal cell density was 10⁴cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

A dose-related inhibition of algal growth rate and yield was found at the two highest test concentrations at the end of the test, resulting in 15 and 54% inhibition, respectively, at the highest test concentration.

Samples taken from all test concentrations and the control were analysed. The measured concentrations were at the level of the nominal concentrations throughout the test, i.e. were at 108-113% relative to the nominal concentrations at the start and at the end of the test. Based on these results, the effect parameters were based on the analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg a.i./L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	<10	32	53	>100	>100
	lower 95%-cl			45
	upper 95%-cl			62
Yield	Value	<10	<10	6.0	14	82
	lower 95%-cl			4.0	11	71
	upper 95%-cl			8.0	17	97

cl – confidence limit; * based on statistical significance;^#based on biological relevance.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Sodium hydrogen substituted amino acid solution inhibited growth rate and yield of this fresh water algae species significantly at analytically confirmed nominal concentrations of 10 mg a.i./L and higher. The 72h-EC₅₀for growth rate inhibition (E_RC₅₀) was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg a.i./L. The 72h-EC₁₀for growth rate inhibition (E_RC₁₀) was 53 mg a.i./L with a confidence interval ranging from 45 mg a.i./L to 62 mg a.i./L.

Description of key information

Based on the results of a short-term aquatic toxicity study, performed according to OECD 201 and GLP principles, Sodium hydrogen substituted amino acid solution inhibited growth of Pseudokirchneriella subcapitata at analytically confirmed nominal concentrations of 10 mg a.i./L and higher. The 72 h-ErC50, ErC10 and NOEC were determined to be >100 mg a.i./L, 53 mg a.i./L and 32 mg a.i./L (based on biological significance), respectively.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

53 mg/L

Additional information

The objective of the study was to evaluate Sodium hydrogen substituted amino acid solution for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata ) during an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀and EC₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011.

The batch of Sodium hydrogen substituted amino acid solution tested was a clear colourless solution with a purity of 35.8% and completely soluble in test medium at the concentrations tested. A correction factor of 2.52 was used to correct for the water content of the test item. Concentrations are expressed as “mg a.i./L” when referring to the water-free fraction of the test item.

A final test was performed based on the results of a preceding range-finding test.Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to nominal concentrations of 10, 18, 32, 56 and 100 mg a.i./L. The initial algal cell density was 10⁴cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

A dose-related inhibition of algal growth rate and yield was found at the two highest test concentrations at the end of the test, resulting in 15 and 54% inhibition, respectively, at the highest test concentration.

Samples taken from all test concentrations and the control were analysed. The measured concentrations were at the level of the nominal concentrations throughout the test, i.e. were at 108-113% relative to the nominal concentrations at the start and at the end of the test. Based on these results, the effect parameters were based on the analytically confirmed nominal concentrations.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg a.i./L)		NOEC*	NOEC^#	EC10	EC20	EC50
Growth rate	Value	<10	32	53	>100	>100
	lower 95%-cl			45
	upper 95%-cl			62
Yield	Value	<10	<10	6.0	14	82
	lower 95%-cl			4.0	11	71
	upper 95%-cl			8.0	17	97

cl – confidence limit; * based on statistical significance;^#based on biological relevance.

In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Sodium hydrogen substituted amino acid solution inhibited growth rate and yield of this fresh water algae species significantly at analytically confirmed nominal concentrations of 10 mg a.i./L and higher. The 72h-EC₅₀for growth rate inhibition (E_RC₅₀) was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg a.i./L. The 72h-EC₁₀ for growth rate inhibition (E_RC₁₀) was 53 mg a.i./L with a confidence interval ranging from 45 mg a.i./L to 62 mg a.i./L.