Tyrosine

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:

From 2017-11-06 to 2017-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tyrosine

EC Number:

200-460-4

EC Name:

Tyrosine

Cas Number:

60-18-4

Molecular formula:

C9H11NO3

IUPAC Name:

tyrosine

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control.
- Sampling method: taken at 0 hours (initial value) from fresh test solutions and after 24 hours, 48 hours and 72 hours from aged solutions from all test item concentrations and control. At each sampling time point a retention sample was taken.
- Sample storage conditions before analysis: stored deep frozen

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: stock solution S1: weighed necessary amount of test item to a volumetric flask and added test medium up to the bench mark, homogenised by shaking. Lower test solutions: dilution of stock solution S1 with test medium
- Evidence of undissolved material: the solution was clear and transparent.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata Hindák
- Strain: SAG 61.81
- Source: commercial supplier
- Age of inoculum (at test initiation):
- Method of cultivation: grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state.

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.7 – 23.1 °C

pH:

7.70 – 8.15

Nominal and measured concentrations:

Nominal: 100, 31.3, 9.77, 3.05, 0.954 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps
- Type: closed
- Fill volume: filled up with ~ 50 mL test solution
- Aeration: CO2 supplied by continuous agitation
- Initial cells density: 0.5 E+04 cells/mL
- Control end cells density: 26.35 E+04 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: AAP-Medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary.
- Photoperiod: Continuously
- Light intensity and quality: fluorescent tubes, 88.8 μEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: fluorescence microplate reader (infinite 200Pro)
- Other: morphological appearance: observed microscopically at the end of the test; pH: Measured at t = 0 h and t = 72 h; temperature: measured continuously and recorded at hours 0, 24, 48 and 72; light intensity: measured once a year and was confirmed for one representative position at test start.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 63.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 63.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

63.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Inhibition: After 72 h at termination of the test no statistically significant inhibition of growth rate or yield was observed for test item concentrations up to and including 100 mg/L. The inhibition of growth rate peaked in 6.6 % and the inhibition of yield peaked in 23.8 % at a nominal test item concentration of 31.3 mg/L.
- Cell morphology: normal for the control and all test item concentrations. At 31.3 and 100 mg/L bacteria were observed in the solutions.
- Analytical results: The measured initial concentration of active ingredient was 81 % of nominal at 100 mg/L. A degradation of the active ingredient was observed during the test with 29 % of nominal measured at test end. The measured initial concentration of active ingredient in the replicates containing the 100 mg/L test solution without algae was 74 % of nominal at 100 mg/L. A degradation of the active ingredient was observed during the test with 46 % of nominal measured at test end.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.23 mg/L for growth rate; 0.685 mg/L for yield; 0.639 mg/L for biomass intergral

Reported statistics and error estimates:

- LOEC and NOEC: Dunnetts-t-test (left-sided, p<0.05) for growth rate and for yield
- ECx: Due to an inhibition below 50% the database was weak for probit analysis which hence was not performed

Any other information on results incl. tables

Toxicological endpoints for the test item:

	Test item [mg/L]
	nominal	Actual (geometric mean)
ErC10 (Growth rate)	>100	>63.2
95% confidential limits	n.c	n.c
ErC20	>100	>63.2
95% confidential limits	n.c.	n.c.
ErC50	>100	>63.2
95% confidential limits	n.c.	n.c.
EyC10 (Yield)	n.d.	n.d.
95% confidential limits	n.c.	n.c.
EyC20	n.d.	n.d.
95% confidential limits	n.c	n.c
EyC50	>100	>63.2
95% confidential limits	n.c.	n.c.
NOEC	100	63.2
LOEC	-	-

n.d.: Not determined

n.c : Not caculated

Individual cell numbers:

Conc. [mg/L]	Cell numbers [1E+04/mL]				Yield
	0h	24h	48h	72h	0h–72h
Control	0.63	1.29	5.63	25.52	24.89
	0.51	1.32	6.05	28.78	28.27
	0.52	1.17	5.02	17.18	16.66
	0.54	1.63	6.07	26.49	25.95
	0.54	1.77	7.57	32.39	31.85
	0.51	1.68	6.73	27.75	27.24
Mean	0.541)	1.48	6.18	26.35	25.81
0.954	0.54	1.01	5.77	25.48	24.94
	0.54	1.65	6.24	25.24	24.70
	0.54	1.75	6.80	25.53	24.99
Mean	0.54	1.47	6.27	25.42	24.88
3.05	0.54	1.18	5.74	20.00	19.46
	0.54	1.74	6.87	29.90	29.36
	0.54	1.62	5.96	22.65	22.11
Mean	0.54	1.51	6.19	24.18	23.64
9.77	0.54	1.43	5.44	21.67	21.13
	0.54	1.61	5.99	25.25	24.71
	0.54	1.49	6.09	25.38	24.84
Mean	0.54	1.51	5.84	24.10	23.56
31.3	0.54	1.13	4.03	17.55	17.01
	0.54	1.46	5.66	18.76	18.22
	0.54	1.23	6.37	24.32	23.78
Mean	0.54	1.27	5.35	20.21	19.67
100	0.54	1.88	6.32	28.60	28.06
	0.54	1.76	7.00	28.50	27.96
	0.54	1.59	7.29	31.67	31.13
Mean	0.54	1.74	6.87	29.59	29.05

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) and 63.2 mg/L (actual) at test end. Thus the overall LOEC was not determinable and the overall NOEC was observed to be at 100 mg/L (nominal) and 63.2 mg/L (actual).
The EC10-, EC20- and EC50-value for growth rate and the EC50-value for yield were considered to be > 100 mg/L (nominal) and > 63.2 mg/L (actual).

Executive summary:

The effects of test item on the growth of the single cell green alga Pseudokirchneriella subcapitata were determined according to OECD 201.

A static test with nominal test item concentrations of 100, 31.3, 9.77, 3.05 and 0.954 mg/L and control was performed. Six replicates were employed for the control and three for each test item concentration. One replicate with 100 mg/L test item concentration without algae was run in parallel which was used for the analytical sampling. The test was performed in 100 mL Erlenmeyer flasks each containing ~ 50 mL test solution.

Toxicological endpoints were evaluated using the nominal and actual concentrations (based on the geometric mean) of the test item.

No statistically significant inhibitory effects on any parameter (growth rate, yield) were observed at any of the test item concentrations, including the highest test item concentration of 100 mg/L (nominal) and 63.2 mg/L (actual) at test end. Thus the overall LOEC was not determinable and the overall NOEC was observed to be at 100 mg/L (nominal) and 63.2 mg/L (actual).

The EC10-, EC20- and EC50-value for growth rate and the EC50-value for yield were considered to be > 100 mg/L (nominal) and > 63.2 mg/L (actual). Due to an inhibition of yield below 50 % and a missing concentration response relation no reliable values were calculable and the EC10- and EC20-value for yield were not determined.