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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 - 14 Aug 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1993

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test chambers at 0 h and 96 h
- Sampling method: collected at mid-depth of vessel
- 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: Individual test solutions were prepared by mixing calculated amounts into 40 L of dilution water and sonicated for approximately 20 minutes and then mixed with top-down electric mixers.
- Differential loading: yes
- Controls: yes, dilution water only
- Evidence of undissolved material: no

Test organisms (species):

Lepomis macrochirus

Details on test organisms:

TEST ORGANISM
- Common name: bluegill
- Source: obtained from Osage Catfisheries, Inc., Osage Beach, USA
- Age: juvenile
- Length at study termination: 2.1 cm (average, n = 10), range: 1.9 - 2.3 cm
- Weight at study termination: 0.09 g (average, n = 10), range: 0.05 - 0.13 g

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: same as test
- Type and amount of food during acclimation: commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, USA
- Feeding frequency during acclimation: daily, no feeding for at least two days prior to the test
- Health during acclimation: No signs of disease or stress and no mortalities occurred during the 7 days prior to the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

138 mg/L as CACO3

Test temperature:

21.5 - 22.8°C

pH:

7.8 - 8.5

Dissolved oxygen:

7.4 - 8.6 mg/L

Nominal and measured concentrations:

Nominal test substance concentrations: 7.5, 15, 30, 60 and 120 mg a.s./L
Mean meas. test substance concentrations: 7.5, 15, 29, 60 and 120 mg a.s./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 54-L aquaria
- Material, size: stainless steel, fill volume: 40 L, depth: 22.5 cm
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.02 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: well water located on the Wildlife International, Ltd. site, passed through sand filter and aerated. Prior to use filtered to 0.45 μm.
- Alkalinity: 181 mg/L as CaCO3 (range: 180 - 182 mg/L as CaCO3)
- Conductivity: 316 μmhos/cm (range: 310 - 320 µmhos/cm)
- pH: 8.2 (range: 8.1 - 8.4)
- Intervals of water quality measurement: temperature, dissolved oxygen and pH: at the beginning of the test and at approximately 24-hour intervals (in each test chamber)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 h day-night cycle
- Light intensity: Fluorescent light, 120 lux

EFFECT PARAMETERS MEASURED: number of dead organisms, individuals exhibiting signs of toxicity or abnormal behavior (6, 24, 48, 72 and 96 h)

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 120 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no sublethal effects observed
- Mortality of control: no

Reported statistics and error estimates:

The absence of mortality in the test precluded the statistical calculation of LC50 values. Therefore, the LC50 values were estimated to be greater than the highest concentration tested.

Sublethal observations / clinical signs:

Table 1: Mortality and sublethal effects after 96 hours.

Mean measured Concentration (mg a.s./L)	No. dead fish	Effects	Cumulative percent mortality
Negative control	0	10 AN	0
7.5	0	10 AN	0
15	0	10 AN	0
29	0	10 AN	0
60	0	10 AN	0
120	0	10 AN	0

AN = Appear normal

Table 2: Validity criteria for OECD 203.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test	0%	yes
The dissolved oxygen concentration must have been at least 60 per cent of the air saturation value throughout the test	> 60%	yes
There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80 per cent of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20 per cent, results should be based on the measured concentration.	Recovery: 96.7 - 100% of nominal	yes

Validity criteria fulfilled:

yes

Remarks:

Please see Table 2 at "Any other information on results and tables" for more details

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 - 11 Dec 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

1993

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all test chambers at 0 h and 96 h
- Sampling method: collected at mid-depth of vessel
- 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: test solution for each test chamber was prepared at a nominal concentration of 120 mg a.s./L by mixing calculated amounts of the test substance into 20 L of dilution water (filtered saltwater) and sonicating for approximately five minutes. The test substance solution was then added to the remainder of the dilution water in the test chamber, the beaker was rinsed with a portion of the dilution water into the test chamber, and the solution was stirred approximately one minute with a steel whisk.
- Differential loading: no
- Controls: yes, dilution water only
- Evidence of undissolved material: no

Test organisms (species):

Cyprinodon variegatus

Details on test organisms:

TEST ORGANISM
- Common name: sheepshead minnow
- Source: obtained from Aquatic BioSystems, Inc., Fort Collins, USA
- Age: juvenile
- Length at study termination: 2.2 cm (average, n = 10), range: 1.9 - 2.5 cm
- Weight at study termination: 0.18 g (average, n = 10), range: 0.13 - 0.26 g

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions: same as test
- Type and amount of food during acclimation: live or frozen brine shrimp nauplii (Artemia sp.) and/or a commerciallyprepared diet supplied by Zeigler Brothers, Inc., Gardners, USA
- Feeding frequency during acclimation: daily, no feeding for at least two days prior to the test
- Health during acclimation: no signs of disease or stress and no mortalities occurred during the 7 days prior to the test.

Test type:

static

Water media type:

saltwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

21.2 - 22.5°C

pH:

7.0 - 8.2

Dissolved oxygen:

7.0 - 7.8 mg/L

Salinity:

20‰

Nominal and measured concentrations:

Nominal test substance concentrations: 120 mg a.s./L
Mean meas. test substance concentrations: 129 mg a.s./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 25-L aquaria
- Material: stainless steel,, fill volume: 20 L, depth: 23.5 cm
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.09 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural seawater collected at Indian River Inlet, Delaware, USA, passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank. The filtered saltwater then was diluted with freshwater from a well on the Wildlife International, Ltd. site and aerated with spray nozzles. The water was filtered to 0.45 µm to remove fine particles prior to its use in the test.
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, dissolved oxygen and pH: at the beginning of the test and at approximately 24-hour intervals (in each test chamber)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 h day-nigh cycle
- Light intensity: Fluorescent light, 155 lux

EFFECT PARAMETERS MEASURED: number of dead organisms, individuals exhibiting signs of toxicity or abnormal behavior (4.5, 24, 48, 72 and 96 h)

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 129 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: no sublethal effects observed
- Mortality of control: no

Reported statistics and error estimates:

The use of a single test concentration precluded the statistical calculation of LC50 values at 24, 48, 72 and 96 hours. Therefore, the LC50 values were estimated to be less than, approximately equal to, or greater than the limit concentration.

Validity criteria fulfilled:

yes

Remarks:

Please see Table 2 at "Any other information on results and tables" for more details

Description of key information

LC50 (96 h) = > 120 mg/L (mean measured, OECD 203, Lepomis macrochirus)

LC50 (96 h) = > 129 mg/L (mean measured, OECD 203, Cyprinodon variegatus)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

120 mg/L

Marine water fish

Marine water fish

Effect concentration:

129 mg/L

Additional information

Two experimental studies testing the acute toxicity of the test substance towards fish are available. Both GLP studies were performed according to OECD 203 in a static sytem. One study tested the effects of the test substance on the freshwater species Bluegill Lepomis macrochirus and the second study was performed as a limit test with the saltwater species Cyprinodon variegatus.

In the key freshwater study, bluegill Lepomis macrochirus was exposed to mean measured concentrations of 7.5, 15, 29, 60 and 120 mg a.s./L and a negative control. The test substance concentrations were were measured directly by high performance liquid chromatography (HPLC) with UV detection and ranged between 98.8 to 101% of nominal concentrations. All bluegill in the negative control and the treatment groups appeared normal throughout the test with no observed mortalities or sublethal effects observed. The 96-hour LC50 value, based on the mean measured concentrations of the test substance, was >120 mg a.s./L, the highest concentration tested.

In the marine key study, sheepshead minnow Cyprinodon variegatus was exposed to a mean, measured concentration of 129 mg a.s./L and a negative control. The targeted test substance concentration was analytically verified via HPLC-UV. The mean measured value was 108% of the nominal concentration. All sheepshead minnows in the negative control group and the 129 mg a.s./L treatment group appeared normal throughout the test with no mortality or sublethal effects observed. The 96-hour LC50 value, based on the mean, measured concentration was >129 mg a.s./L, the only concentration tested.