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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Apr - 01 May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

1998

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all treatments at test initiation, at the beginning and the end of the longest renewal cycle each week, and at test termination
- Sampling method: from mid-depth in the test solutions
- Sample storage conditions before analysis: processed immediately

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test concentration preparation three times per week during the first two weeks of the test, and twice during the last week of the test. A primary stock solution was prepared in dilution water at a nominal concentration of 50 mg a.s./L and sonicated for five minutes. Proportional dilutions of the primary stock solution were made in dilution water to prepare test solutions.
- Differential loading: no
- Controls: yes, dilution water only
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: < 24 h
- Method of breeding: same conditions as in test
- Source: bred at testing facility

Feeding during test
- Food type: mixture of yeast, cereal grass media, and trout chow and a suspension of the freshwater green alga, Pseudokirchneriella subcapitata
- Frequency: once daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

132 - 148 mg/L as CaCO3

Test temperature:

19.1 - 20.6 °C

pH:

8.1 - 8.4

Dissolved oxygen:

7.8 - 8.9 mg/L

Conductivity:

315 - 361 µmhos/cm

Nominal and measured concentrations:

Nominal test substance concentrations: 0.38, 0.75, 1.5, 3.0 and 6.0 mg a.s./L
Mean meas. test substance concentrations: 0.37, 0.73, 1.5, 3.0 and 6.0 mg a.s./L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass beakers
- Type: loosely covered with a plastic petri dish
- Fill volume: 220 mL
- Headspace: 30 mL
- Aeration: no
- Renewal rate of test solution: three times per week in week 1 and 2, twice in week 3
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20

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 and passed through an ultraviolet sterilizer
- Total organic carbon:< 2 mg C/L
- Alkalinity: 182 mg/L as CaCO3 (range: 180 - 186 mg/L as CaCO3)
- Conductivity: 303 μmhos/cm (range: 300 - 310 µmhos/cm)
- pH: 8.2 (range: 8.1 - 8.3)
- Intervals of water quality measurement: Temperature, dissolved oxygen and pH: at day 0, and at day 21 in the old and new solutions on renewal days (in two alternating replicate test chambers of each treatment and control group), hardness, alkalinity and specific conductance: at day 0, 8 and 15 (negative control and the highest test concentration) and at day 21 (pooled replicate solutions at test termination)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 h day-night cycle
- Light intensity: 235 lux (Fluorescent light)

EFFECT PARAMETERS MEASURED: immobility, clinical signs of toxicity, presence of eggs in the brood pouch, aborted eggs, males or ephippia: daily, neonates produced (three times per week and at day 21), body length and dry weight of each surviving first-generation daphnid (day 21)

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

adult survival/growth

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks:

immobility

Details on results:

- Observations on body mean length and weight: negative control: 5.3 mm (length), 1.55 mg (dry weight), 0.37, 0.73, 1.5, 3.0 and 6.0 mg a.s./L treatment groups: 5.1, 5.4, 5.4, 5.3 and 5.4 mm (length), 1.53, 1.71, 1.72, 1.65 and 1.64 mg (dry weight)
- Mortality of control: 10%

Reported statistics and error estimates:

Discrete-variable data were analyzed using Chi-square and Fisher’s Exact test to identify treatment groups that showed a statistically significant difference (p ≤ 0.05) from the control. All continuous-variable data were evaluated for normality using the Shapiro-Wilk or Chi-Square test and for homogeneity of variance using Levene’s test (p = 0.01). The reproduction and dry weight data passed the assumptions of normality and homogeneity. Analysis of variance (ANOVA) was used to determine whether or not statistically significant differences existed among the experimental groups (p = 0.05). Those treatments that were significantly different from the control means were identified using either Bonferroni’s t-test or Dunnett’s test (p ≤ 0.05). All statistical tests were performed using a personal computer with TOXSTAT or SAS software.

Table 1: Summary of effects following exposure of Daphnia magna for 21 days

Mean, measured technical concentration (mg a.s./L)	Mean % adult survival a)	Mean first day of reproduction b)	Mean total live young c)	Mean total immobile young d)	Mean adult length (mm) e)	Mean adult dry weight (mg) e)
Water Control (0.0)	90	7	296	0	5.3	1.55
0.37	60	7	285	0	5.1	1.53
0.73	70	7	306	0	5.4	1.71
1.5	60	7	305	0	5.4	1.72
3.0	90	7	292	0	5.3	1.65
6.0	90	7	310	2	5.4	1.64

a) Percent of adult daphnids alive at the end of the test (immobility was synonymous with death)

b) First day that reproduction was observed in the replicates

c) Mean of live young produced per surviving female

d) Mean of immobile young produced per surviving female; calculated from 14 immobile neonates, of 98 neonates, counted for Replicate I on Day 17

e) There were no significant differences from the control (adult length and dry weight: Dunnett’s t-test, alpha = 0.05)

Table 2: Validity criteria for OECD 211.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The mortality of the parent animals in the control (female Daphnia) does not exceed 20% at the end of the test.	10%	yes
The mean number of living offspring produced per parent animalsurvivingat the end of the test is ≥ 60 in the control.	296	yes

Validity criteria fulfilled:

yes

Remarks:

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

Description of key information

NOEC (21 d) = ≥ 6.0 mg a.s./L (mean measured, OECD 211, Daphnia magna)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

6 mg/L

Additional information

One experimental GLP study is available testing the chronic toxicity towards aquatic invertebrates. This study was conducted according to OECD 211 with water flea Daphnia magna.

In the semi-static experiment, daphnids were exposed to mean measured concentrations between 0.37 and 6.0 mg a.s./L for 21 days. The test solutions were renewed three times per week. The targeted test substance concentrations were analytically measured by high
performance liquid chromatography (HPLC) with UV detection. The mean measured ranged from 97 to 100% of the targeted nominal concentrations. The daphnids showed no significant reduction in survival rate, reproduction or growth at any of the concentrations tested. Consequently, the NOEC (21 d) was ≥ 6.0 mg a.s./L (mean measured).