Prometryn

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jun 2000 to 16 Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples of each test concentration and stock solution were drawn from the approximate center of the vessels. They were taken immediately before the exposure and after 7, 14, 21 and 28 days exposure. An additional specimen of the highest concentration was taken on day 24, when all the fish had died in this concentration. The specimens were either analyzed immediately or kept at -18 °C to -22 °C until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- 3.54 to 3.55 g of the test item were added to water and made up to 150 L with water on Days 5, 3, 8, 16 and 24.
- 2.13 to 2.15 mg of the test item were added and made up to 90 L with water on Days 0, 13 and 21.
- 1.18 g of the test item were added and made up to 50 L with water on Days 1, 2, 7 and 12.
- 827 to 830 mg of the test item were added to water and made up to 35 L with water on Days 15, 20 and 23.

The stock solution was pumped into the mixing chamber of a modified Mount and Brungs (1967) proportional diluter to produce the desired test concentrations. After 5 days of operation of the dosing system, the fish were impartially placed into the aquaria (beginning of exposure).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow Trout
- Length at study initiation: mean length of 49 mm (range: 46 - 52 mm) based on 10 fish of the blank control on Day 1
- Weight at study initiation: mean weight of 1.0 g (range: 0.9 - 1.2 g) based on all 70 fish on Day 1
- Handling: Fine-mesh dip net were used to transfer fish, taking care to minimise possible stress due to handling

ACCLIMATION
- Acclimation period: 13 days
- Acclimation water was similar quality as the water used in the test
- Type and amount of food: Commercially prepared trout food HOKOVIT No. 502 supplemented by living, frozen or dried organisms, e.g. daphnia and larvae of mosquitoes, until 24 hours prior to the test. 8 days before the start of the test, the amount of food was reduced to 1.5 % of the body weight.
- Fish were not fed 24 hours prior to the start of the test.

FEEDING DURING TEST
The fish were fed a commercially prepared diet HOKOVIT No. 502, 4 % of initial body weight daily except on Sundays, given in two feeds separated by at least 5 hours (morning / afternoon). Fish were not fed on day 28 of the exposure.

PRETREATMENT
To obtain maximum sensitivity of the growth response as a toxicity endpoint, individual fish were freeze branded one day before the start of the exposure after light anaesthesia with 0.10 g/L tricaine methane sulphonate (MS 222). Fish were weighed and measured during anaesthesia. The fish were allocated at random for 24h into recovering tanks, and then transfered directly as groups into the test tanks.

TREATMENT
Fish were not treated against diseases during holding.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

- Control: 174 mg/L as CaCO3
- Highest concentration: 182 mg/L as CaCO3

Test temperature:

13.0 - 13.9 °C

pH:

8.1 - 8.4

Dissolved oxygen:

79 - 101% of the saturation

Nominal and measured concentrations:

Nominal concentration: 0.15, 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L
Mean measured concentration: 0.12, 0.25, 0.52, 0.99, 2.0 and 4.4 mg/L, respectively. See Table 1 in "Any other information on material and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 20 L glass aquaria
- Filled volume: 15 L of water
- Size of vessel: 35 cm x 22 cm x 26 cm (depth of water: 20 cm)
- Front of the vessel was covered to minimize disturbance of fish.
- Type of flow-through: proportional diluter
- Flow rate: 6 tank volumes/day (flow rate of 30 L/h (4 L/h and tank) with deviations in the range of -5 to -3%
- No. of organisms per vessel: 10
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: 0.12 g of fish/L in 24 hours

DILUTION WATER
- Source/preparation of dilution water: Well water of ’Lange Erlen' and 'Hard' was used as dilution water. The water in the inhouse storage tank was aerated with spray nozzles. Prior to its use, the water passed through an activated carbon filter and a UV sterilizer.
- Hardness: 150 - 180 mg/L as CaCO3
- Alkalinity: 284 - 320 mg/L as CaCO3
- pH: 6.6 - 8.0
- Dissolved oxygen: 8.8 mg/L
- Conductivity: 261 - 443 µmhos/cm

WATER PARAMETER
The pH6, temperature (°C) and oxygen content (mg/L) were measured on Mondays, Wednesdays and Fridays in each test chamber. In addition, the temperature was measured continuously in one test chamber. Total hardness (as CaC03) and alkalinity was measured in the blank control chamber and in the highest concentration at the start of the test. Characteristics of the test solutions (such as precipitated materials, cloudiness, etc.) were recorded in daily intervals except on Sundays.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours/8 hours (light/dark) with 30 min transition period
- Light source: Fluorescent light

EFFECT PARAMETERS MEASURED
Observations of sublethal symptoms, such as abnormal behavioural activity and stress (swimming behaviour, loss of equilibrium, respiratory function, exophthalmus, pigmentation, feeding activity, reaction to external stimuli and other observations) as well as mortality were made daily, except at the starting day and on most Sundays (days 0, 4, 11, 18 and 25, respectively). Dead fish, if any, were removed from the test solutions at least at the above mentioned intervals. The weight of the fish was measured at the start (day - 1) and at the end of exposure, together with the length of the control fish.

RANGE-FINDING STUDY
- Based on the results of the acute test with Rainbow Trout yielding an LC50 (96h) of 5.5 mg/L, the following nominal concentrations were selected: 0.15, 0.30, 0.60, 1.2, 2.4, 4.8 mg/L.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

interms of weight

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.52 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks:

in terms of weight

Details on results:

Overview of the results were provided in “Any other information on results incl. tables”

LETHAL EFFECTS
At mean measured concentrations of 0.52, 0.99, 2.0 and 4.4 mg/L, mortalities at the end of the exposure were, 10, 0, 30 and 100%, respectively. The 10% mortality observed at 0.52 mg/L was not caused by the test substance and is within the 10% mortality tolerated in the control for the test to be valid. The NOEC (28d) corresponded to 2.0 mg/L, since there was no statistically significant difference in relation to the control. The mortality in the blank control was 0 %.

SUBLETHAL EFFECTS
After 28 d exposure, the average weight of the fish in the blank control had increased by 223% (calculated by weight at end/ weight at start *100). At a concentration of 0.25 mg/L, no statistically significant effects were found for the usually most sensitive growth parameter (individual fish specific growth rates in terms of weight). In addition, no statistically significant effects were found in regard to other sublethal effects. Thus, an NOEC (28d) of 0.25 mg/L resulted . Endpoints, such as change in swimming behaviour and in pigmentation, as well as reactions to external stimuli and feeding activity turned out to be the next most sensitive parameters showing initial effects decreasing over time and yielding an NOEC (28d) of 0.52 mg/L.
The logit analysis yielded estimated EC-values in regard to the individual fish specific growth rate in terms of weight over the period of the test of EC10 (28d) equaling 0.47 mg/L and EC20 (28d) equaling 0.49 mg/L respectively.

Reported statistics and error estimates:

Procedures Used
Results of the toxicity test were interpreted by standard statistical techniques using SAS Version 6.11. and the procedure ‘ECOS’ by Fisch and Strehlau, 1998 using SAS PROC GLM and PROC NLIN

Significance Level
All tests were performed using a significance level of α = 0.05.

Dichotomous Data
For the determination of NOECs in regard to dichotomous data, the procedure ‘ECOS’ was used which performed the Fisher Exact Test. The
number of affected fish (lethal and non-lethal effects) divided by the number of living fish per concentration were used as experimental units.

Continuous Data
For the determination of NOECs in regard to continuous data, the procedure ‘ECOS’ was used which performed Dunnett’s multiple means comparison test (Dunnett, 1955 & 1980) using mean values per concentration of the instantaneous individual growth rates as experimental units.
For this purpose, the weight of the fish at the start (day-1) WT1 and at the end of the exposure WT2 were transformed to obtain:
Individual fish specific growth rate n in terms of weight:
RWEIGHT = 1 /days * In (WT2/WT1)
Bartlett’s Test (Bartlett, 1937) was used to check for the constancy of variance, and a t-test to compare the blank and the vehicle control, if present.

Table 2. Weight and growth rate results

Measured concentration (mg/L)	Rweight^a	WT1^b	WT2^c		Measured concentration (mg/L)	Rweight	WT1	WT2
B	0.026343	1.1	2.3		0.99	-0.018244	1.0	0.6
B	0.031267	1.0	2.4		0.99	-0.007969	1.0	0.8
B	0.028962	1.2	2.7		0.99	-0.011373	1.1	0.8
B	0.026213	1.2	2.5		0.99	-0.014481	0.9	0.6
B	0.022923	1.0	1.9		0.99	-0.010274	1.2	0.9
B	0.038896	1.2	3.1		0.99	-0.006511	1.2	1.0
B	0.028518	0.9	2.0		0.99	-0.007969	1.2	1.5
B	0.026343	1.1	2.3		0.99	-0.008976	0.9	0.7
B	0.030261	0.9	2.1		0.99	-0.011373	1.1	0.8
B	0.029747	1.0	2.3		0.99	-0.008976	0.9	0.7
0.12	0.026213	1.2	2.5		2.0	-0.006511	1.2	1.0
0.12	0.026686	0.9	1.9		2.0	-0.012738	1.0	0.7
0.12	0.031922	0.9	2.2		2.0	-0.007167	1.1	0.9
0.12	0.024755	0.9	1.8		2.0	-0.012738	1.0	0.7
0.12	0.024755	1.1	2.2		2.0	-	0.9	-
0.12	0.031922	0.9	2.2		2.0	-0.011373	1.1	0.8
0.12	0.026498	1.0	2.1		2.0	-0.012738	1.0	0.7
0.12	0.026213	1.2	2.5		2.0	-0.012738	1.0	0.7
0.12	0.029321	1.1	2.5		2.0	-	0.9	-
0.12	0.028518	0.9	2.0		2.0	-	1.0	-
0.25	0.028518	0.9	2.0		4.4	-	1.2	-
0.25	0.017588	1.1	1.8		4.4	-	1.1	-
0.25	0.028159	1.0	2.2		4.4	-	1.1	-
0.25	0.026213	1.2	2.5		4.4	-	1.0	-
0.25	0.036488	0.9	2.5		4.4	-	1.0	-
0.25	0.018951	1.0	1.7		4.4	-	1.2	-
0.25	0.014481	1.0	1.5		4.4	-	0.9	-
0.25	0.026213	1.2	2.5		4.4	-	1.2	-
0.25	0.018951	1.0	1.7		4.4	-	1.0	-
0.25	0.024755	0.9	1.8		4.4	-	1.0	-
0.52	0.003404	1.1	1.1
0.52	0.007969	1.2	1.5
0.52	0.010274	1.2	1.6
0.52	0.018244	1.2	2.0
0.52	0.006511	1.0	1.2
0.52	0.017588	1.1	1.8
0.52	0.023094	1.1	2.1
0.52	-	1.0	-
0.52	0.019519	1.1	1.9
0.52	0.006511	1.0	1.2

a. Individual fish specific growth rate r_I in terms of weight

b. Individual weight of fish in g at the start of exposure (day-1)

c. Individual weight of fish in g at the end of exposure (day 28)

Table 3. Summary of NOEC/LOEC

Based on measured mean concentration	NOEC (mg/L)	LOEC (mg/L)
Lethal effect: Mortality	2.0	1.4
Non-lethal effects: growth rate (Individual fish specific growth rate in terms of weight)	0.25	0.52
Other observed non-lethal effects:
Change in swimming behavior	0.52	0.99
Loss of equilibrium	0.99	2.0
Change in respiratory movement	4.4	>4.4
Exophthalmus	4.4	>4.4
Change in pigmentation	0.52	0.99
Feeding activity	0.52	0.99
Reaction to external stimuli	0.52	0.99
	EC10 (mg/L)	EC20 (mg/L)
Growth rate (Individual fish specific growth rate in terms of weight)	0.47	0.49

Validity criteria fulfilled:

yes

Conclusions:

Based on the mean measured concentrations and effects on growth (weight), the NOEC for the test substance was determined to be 0.25 mg/L and LOEC was determined to be 0.52 mg/L.

Executive summary:

The chronic effect of continuous exposure to the test substance on fish was evaluated in a flow-through juvenile growth toxicity test with the freshwater rainbow trout, Oncorhynchus mykiss. The test was performed according to OECD 215 guideline and in compliance with GLP. The effect assessment of this 28-day exposure test was based on sublethal effects, such as growth and other observed symptoms, and mortality. The fish were exposed to the mean measured concentrations of 0.12, 0.25, 0.52, 0.99, 2.0 and 4.4 mg/L of the test substance (measured by HPLC, nominal concentrations were 0.15, 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L). A negative control was involved in this test as well. The stock solution (an appropriate amount of test substance dissolved in an appropriate volume of test water without using a solvent) was pumped into the mixing chamber of a modified Mount and Brungs (1967) proportional diluter to produce the desired test concentrations. The flow of water to the diluter was 30 L/h (4 L/h and tank) with deviations in the range from -5 to -3%. After 5 days of operation of the dosing system, the fish (10 fish per test group) were randomly placed into the aquaria (35/22/26 cm; depth of the water: 20 cm) expressing the beginning of exposure. Test conditions: pH 8.1 – 8.4, 13.0 - 13.9 °C, dissolved oxygen 79 - 101% of the saturation level, total hardness 174 mg CaCO3/L in the blank control and 182 mg/L in the highest concentration, alkalinity 3.5 mmol/L in the control and 3.5 mmol/L in the highest concentration. The test item appeared homogeneously distributed in the test vessels at all test times and test concentrations. No precipitation was observed at any of the concentrations throughout the period of the exposure. 

 

At mean measured concentrations of 0.12, 0.52, 0.99, 2.0 and 4.4 mg/L, mortalities at the end of the exposure were 0, 10, 0, 30 and 100%, respectively. The 10% mortality observed at 0.52 mg/L is considered not to be caused by the test substance due to missing effect at the next higher test concentration, and, furthermore, it is within the 10% mortality tolerated in the control for the test to be valid. Hence, the NOEC (28d) for lethal effects corresponded to 2.0 mg/L, since there was no statistically significant difference in relation to the control showing a mortality of 0%. After 28 days of exposure, the average weight of the fish in the blank control had increased by 223%. At the concentrations of 0.12 and 0.25 mg/L, no statistically significant effects were found for this growth parameter (individual fish specific growth rates in terms of weight), just as no other statistically significant sublethal effects were found in both treatment groups. For all higher treatment groups, development of body weight was significantly decreased in comparison with the control group by the end of the test period. Thus, an overall NOEC (28d) of 0.25 mg/L resulted and, the LOEC (28 days) was 0.52 mg/L. The corresponding 28-day EC10 and EC20 values were 0.47 and 0.49 mg/L for the individual fish specific growth rate in terms of weight, respectively.

Description of key information

All available data were assessed and the study representing the worst-case effects is included here as key. The results can be considered worst-case and are selected for the CSA. Other studies are included as supporting information.

Freshwater 28-d NOEC = 0.25 mg/L, Oncorhynchus mykiss, growth, OECD TG 215, Rufli 2000

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

0.25 mg/L

Additional information

Freshwater

The chronic effect of continuous exposure to the test substance on fish was evaluated in a flow-through juvenile growth toxicity test with the freshwater rainbow trout, Oncorhynchus mykiss. The test was performed according to OECD 215 guideline and in compliance with GLP. The effect assessment of this 28-day exposure test was based on sublethal effects, such as growth and other observed symptoms, and mortality. The fish were exposed to the mean measured concentrations of 0.12, 0.25, 0.52, 0.99, 2.0 and 4.4 mg/L of the test substance (measured by HPLC, nominal concentrations were 0.15, 0.30, 0.60, 1.2, 2.4 and 4.8 mg/L). A negative control was involved in this test as well. The stock solution (an appropriate amount of test substance dissolved in an appropriate volume of test water without using a solvent) was pumped into the mixing chamber of a modified Mount and Brungs (1967) proportional diluter to produce the desired test concentrations. The flow of water to the diluter was 30 L/h (4 L/h and tank) with deviations in the range from -5 to -3%. After 5 days of operation of the dosing system, the fish (10 fish per test group) were randomly placed into the aquaria (35/22/26 cm; depth of the water: 20 cm) expressing the beginning of exposure. Test conditions: pH 8.1 – 8.4, 13.0 - 13.9 °C, dissolved oxygen 79 - 101% of the saturation level, total hardness 174 mg CaCO3/L in the blank control and 182 mg/L in the highest concentration, alkalinity 3.5 mmol/L in the control and 3.5 mmol/L in the highest concentration. The test item appeared homogeneously distributed in the test vessels at all test times and test concentrations. No precipitation was observed at any of the concentrations throughout the period of the exposure. At mean measured concentrations of 0.12, 0.52, 0.99, 2.0 and 4.4 mg/L, mortalities at the end of the exposure were 0, 10, 0, 30 and 100%, respectively. The 10% mortality observed at 0.52 mg/L is considered not to be caused by the test substance due to missing effect at the next higher test concentration, and, furthermore, it is within the 10% mortality tolerated in the control for the test to be valid. Hence, the NOEC (28d) for lethal effects corresponded to 2.0 mg/L, since there was no statistically significant difference in relation to the control showing a mortality of 0%. After 28 days of exposure, the average weight of the fish in the blank control had increased by 223%. At the concentrations of 0.12 and 0.25 mg/L, no statistically significant effects were found for this growth parameter (individual fish specific growth rates in terms of weight), just as no other statistically significant sublethal effects were found in both treatment groups. For all higher treatment groups, development of body weight was significantly decreased in comparison with the control group by the end of the test period. Thus, an overall NOEC (28d) of 0.25 mg/L resulted and, the LOEC (28 days) was 0.52 mg/L. The corresponding 28-day EC10 and EC20 values were 0.47 and 0.49 mg/L for the individual fish specific growth rate in terms of weight, respectively.