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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jan - 28 Apr 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

1992

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: collected from two replicate test chambers of each control and treatment group two days prior to the start of the test, from two alternating replicates of each control and treatment group at test initiation, at approximately weekly intervals during the test, and at test termination
- 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: pretest period and the first eight days of test: six liter primary stock solution of 3.0 mg a.s./L, primary stock solution: three, two-liter stocks and stirring the stocks together. Each two-liter stock was sonicated and mixed by inversion. Proportional dilutions of the primary stock to prepare four additional stock solutions at nominal concentrations of 0.188, 0.375, 0.75 and 1.5 mg a.s./mL. Stock solutions were delivered to the diluter mixing chambers and mixed with dilution water to achieve the desired nominal test concentrations. Beginning on Day 8: primary stock solution concentration was changed to 1.5 mg a.s./mL. primary stock solution was prepared by making five to ten, two-liter stocks and stirring the stocks together. Each two-liter stock was sonicated and mixed by inversion. Proportional dilutions of the primary stock were made to prepare four additional stock solutions at nominal concentrations of 0.094, 0.188, 0.375 and 0.75 mg a.s./mL.
- Differential loading: no
- Controls: dilution water only
- Evidence of undissolved material: test solutions in the test chambers for all treatment and control groups appeared clear and colorless

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbow trout
- Source: unfertilized eggs and sperm were obtained from Troutlodge, Inc., Sumner, Washington, USA

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): Gametes from at least 3 females and 3 males
- Subsequent handling of eggs, embryos and larvae: eggs were fertilized in the labortatory and the test was initiated within 24 hours of fertilization.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

90 d

Hardness:

132 - 144 mg CaCO3/L

Test temperature:

11.6 - 12.8°C

pH:

8.0 - 8.3

Dissolved oxygen:

7.8 - 9.7 mg/L

Conductivity:

260 - 275 µmhos/cm

Nominal and measured concentrations:

Nominal test substance concentrations: 0.75, 1.5, 3.0, 6.0 and 12 mg a.s./L
Mean meas. test substance concentrations: 0.69, 1.5, 2.9, 5.8 and 11 mg a.s./L

Details on test conditions:

TEST SYSTEM
- Emybro cups: glass cylinders (55 mm diameter, 425-µm nylon screen mesh attached to the bottom with silicone sealant.)
- Test vessel: 9 L aquaria
- Material: glass, fill volume: 7 L, depth: 15.5 cm
- Aeration: no aeration for the first 70 days
- Type of flow-through: continuous-flow diluter with a peristaltic pump (Cole-Parmer Instrument Company, Chicago, Illinois), calibrated prior to the test
- Renewal rate of test solution: 6 volume additions of test water in each test chamber/day
- No. of fertilized eggs/embryos per vessel: 15/cup, 30/treatment group, when more than 90% of the negative control larvae reached the swim-up stage on Day 17 post-hatch, the number of larvae in all replicates was reduced to 15 to prevent overcrowding.
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Fertilization control: 30 embryos, 4 replicates, sacrificed on day 12 to evaluate the fertilization success
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: 0.38 g fish/L/day (end of test), instantaneous loading: 3.9 g fish/L of test water in the test chamber at any given time
- Feeding: salmon-starter mash (Zeigler Brothers, Inc., Gardners, USA) from day 42, three times per day on weekdays and at least two times daily on weekends and holidays, no feeding 51 hours prior to the termination of the test

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 was passed through an ultraviolet sterilizer
- Intervals of water quality measurement: temperature (in each test chamber at the beginning and end of the test and at weekly intervals), dissolved oxygen (in alternating replicates of each treatment and control group at test initiation, daily during the first seven days of the test, at least weekly during the test, and at test termination), pH (from alternating replicates of each treatment and control group at test initiation, at weekly intervals during the test, and at test termination), hardness, alkalinity and specific conductance (alternating replicates of the negative control and the highest surviving concentration treatment group at test initiation, at weekly intervals during the test, and at test termination)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: The rainbow trout embryos and larvae were kept in darkness except during observations until one week after hatching. 16:8 h day-night cycle
- Light intensity: 79 lux

EFFECT PARAMETERS MEASURED: embryo survival (hatching success), time to hatch, time for larvae to swim-up, and post-hatch survival and growth; length of each fish (at 30 days post-hatch, test end), wet weight, dry weight and total length of each surviving fish (test end)

Reference substance (positive control):

no

Duration:

90 d

Dose descriptor:

NOEC

Effect conc.:

11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: time to hatch, hatching success, time to swim-up, survival and growth

Remarks on result:

other: highest test concentration

Details on results:

- Hatching success: averaged 95% (negative control group), 0.69, 1.5, 2.9, 5.8 and 11 mg a.s./L treatment groups: 96, 91, 92, 93 and 93%
- Survival from post-hatch to thinning: control: 98%, treatment groups: ≥ 99%
- Days to hatch and numbers hatched: hatching start: day 23, all surviving embryos in the control and treatment groups had hatched by Day 26
- Egg viabilty in fertilization control: 98%
- Type of and number with behavioural abnormalities: One larva in the 5.8 mg a.s./L treatment group was noted with a curved spine. This isolated observation was not considered to be treatment-related. One fish in Replicate C of the negative control group was noted swimming erratically at the end of Week 3 post-hatch. A mortality, presumably the fish noted swimming erratically, was noted in Replicate C of the control group the following day. One fish in the 5.8 mg a.s./L treatment group was noted as missing and presumed dead.
- Data for length and weight of surviving fish on 65 dph: control: 57.5 mm, 0.69, 1.5, 2.9, 5.8 and 11 mg a.s./L treatment groups: 57.2, 58.0, 57.5, 57.8 and 58.1 mm, respectively

Reported statistics and error estimates:

Continuous variable data (total length, wet and dry weight) were evaluated for normality using the Chi-square test and for homogeneity of variance using Levene’s test (p = 0.01). In each case, the data passed the assumptions of normality and homogeneity and, therefore, analysis of variance (ANOVA) and Dunnett’s t-test were used to evaluate differences between the treatment and negative control means (p = 0.05). Discrete-variable data (hatching success, time to swim-up and survival) were analyzed using Chi-square and Fischer’s Exact test to identify treatment groups that showed a statistically significant difference (p ≤ 0.05) from the negative control. All statistical tests were performed using a personal computer with TOXSTAT or SAS.

Table 1: Summary of effects following exposure of Rainbow Trout to DPX-MAT28 technical for 90 days

 

Mean, measured DPX-MAT28 technical concentration (mg a.s./L)	Mean % hatch a,e	% Swim-up b,e	Mean % survival from hatch to swim-up c,e	Mean % survival from thinning to term d,e	Mean term length (mm) e	Mean term dry weight (g) e
Water Control (0.0)	95	94	98	98	57.5	0.39
0.69	96	91	99	100	57.2	0.39
1.5	91	91	99	100	58.0	0.41
2.9	92	96	99	100	57.5	0.39
5.8	93	95	99	98	57.8	0.40
11	93	97	100	100	58.1	0.41

a: Percent hatched of live embryos

b: Percent larvae to reach swim-up stage prior to thinning on Day 17 post hatch.

c: Percent larvae to survive to thinning on Day 17 post hatch.

d: Percent larvae to survive from thinning on Day 17 post hatch to test termination.

e: There were no significant differences from the control (Percent hatch, swim-up and survival: Fisher’s Exact Test, alpha = 0.05; and length and dry weight: Dunnett’s t-test, alpha = 0.05).

Table 2: Validity criteria for OECD 210.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The dissolved oxygen concentration should be >60% of the air saturation value throughout the test.	>60%	yes
The water temperature should not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Annex 2).	11.6 - 12.8°C	yes
The analytical measure of the test concentrations is compulsory.	Analytical measurements were performed.	yes
Overall survival of fertilised eggs and post-hatch success in the controls and, where relevant, in the solvent controls should be greater than or equal to the limits defined in Annex 2.	Hatching succes and post-hatch success > 75%	yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

NOEC (90 d) = 11 mg/L (mean measured, OECD 2010, Oncorhynchus mykiss)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

11 mg/L

Additional information

One experimental GLP study testing the long-term toxicity of the test substance towards fish is available.

Rainbow trout (Oncorhynchus mykiss) were exposed to nominal concentrations between 0.75 and 12 mg a.s./L under flow-through conditions for 90 days according to OECD 210. Test substance concentrations were analytically verified via high performance liquid chromatography (HPLC) with UV detection. The mean, measured values ranged from 92 to 100% of the targeted nominal concentrations.
There were no treatment-related effects on hatching success, time to hatch, time for larvae to swim-up, or post-hatch growth and survival. The NOEC, based on the time to hatch, hatching success, time to swim-up, survival and growth was 11 mg a.s./L (mean measured). The LOEC was >11 mg a.s./L, the highest concentration tested.