6-Amino-5-chloro-2-cyclopyrimidine-4-carboxylic 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:

04 - 07 Jan 2008

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:

2006

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test concentrations
- Sampling method: at test start and after 72 h (72 h samples were pooled)
- Sample storage conditions before analysis: processed immediately

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The highest test concentration was prepared by mixing the required amount of test substance with OECD freshwater algal medium. The test concentrations were prepared separately by dilution of the stock solution.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source (laboratory, culture collection): maintained at testing facility, originally obtained from University of Toronto, USA

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Acclimation period: at least 14 d

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.4 - 23.9 °C

pH:

test start: 6.1 - 7.9
test end: 6.0 - 8.2

Nominal and measured concentrations:

Nominal test substance concentrations: 7.5, 15, 30, 60 and 120 mg/L
Mean measured test substance concentrations: 7.4, 15, 30, 59 and 120 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass 250 mL Erlenmeyer flasks
- Type: plugged with foam stoppers
- Fill volume: 100 mL
- Headspace: 150 mL
- Aeration: no but shaken continuously at 100 rpm
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD freshwater algal medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD freshwater algal medium
- Culture medium different from test medium: same as test

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continous illumination
- Light intensity and quality: cool-white fluorescent lighting at 5030 to 7100 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:after 0, 24, 48 and 72 h (hemacytometer, microscope)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

7.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 7.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Reported statistics and error estimates:

The cell density, area under the growth curve, and growth rate data were evaluated for normality and homogeneity of variance (α = 0.01) using the Shapiro-Wilk’s and Levene’s tests, respectively. Since the data were normally distributed with homogeneous variances, the treatment groups were compared to the blank control using ANOVA and Dunnett’s test (α = 0.05). Due to high variability in the test, Dunnett’s test was not considered to be sensitive for the determination of the NOEC. Therefore, the results were also analyzed using a Jonckheere-Terpstra trend test (α = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC and LOEC relative to each parameter at 72 hours. (SAS Version 8.2 (4))

Table 1. Summary of algal growth inhibition

Mean, measured concentration [mg/L]	% Inhibition relative to untreated control [growth rate]
0	—
7.4	1
15	1*
30	3*
59	2*
120	12*

  * significantly different from the control (Jonckheere-Terpstra, alpha = 0.05)

Table 2: Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	>16	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	21%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.7%	yes

 

Validity criteria fulfilled:

yes

Remarks:

Please see table 2 at "Any other information on results incl. tables" for more details

Description of key information

ErC50 (72 h) = > 120 mg/L (mean measured, OECD 201, Pseudokirchneriella subcapitata)

NOErC (72 h) = 7.4 mg/L (mean measured, OECD 201, Pseudokirchneriella subcapitata)

Key value for chemical safety assessment

EC50 for freshwater algae:

120 mg/L

EC10 or NOEC for freshwater algae:

7.4 mg/L

Additional information

One experimental GLP study conducted according to OECD 201 is available testing the toxicity towards freshwater algae.

In this study freshwater alga, Pseudokirchneriella subcapitata, was exposed according to OECD 201 to mean measured concentrations in the range of 7.4 and 120 mg/L. Test substance concentrations were measured directly by high performance liquid chromatography (HPLC) with UV detection. Mean measured concentrations ranged from 98 to 100% of the targeted nominal concentrations. After 72 hours, algae were evaluated for effects on cell density, and growth rate.

EC50 values were determined to be > 120 mg a.s./L (mean measured) for growth rate. A Jonckheere-Terpstra test indicated that the 72-hour NOEC and LOEC were determined to be 7.4 mg a.s./L and 15 mg a.s./L (mean measured), respectively, for cell density and growth rate.