Urea

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Deviations:

no

Remarks:

fish species

Principles of method if other than guideline:

- Principle of test: juvenile growth test
- Short description of test conditions:
Sublethal effects of urea on food intake, growth and conversion efficiency were studied in O. mossambicus rearing them individually in plastic trough (33 cm in diameter, 20 cm depth) with different concentrations of urea (10,000, 14,000, 18,000, and 20,000 mg L−1). Five individuals were reared in each concentration and control animal were maintained in fertilizer free medium. The fishes were fed on known amount of fresh goat liver pieces once a day for an hour (10–11 a.m.). The fresh liver was dried in a hot air oven for 24 h to weight consistency and the water content of the food was estimated. The unfed remains were carefully collected using a pipette without causing disturbance to the fish. The rearing medium was changed daily.The feaces were collected by filtering the medium while changing the water and dried at 80 ◦C. The fishes were maintained in this media for 28 days at 28±1 ◦C with normal light.
After 28 days of experimental period, the test fishes were starved for a day to ensure complete evacuation of the gut. They were weighed alive and then killed. The killed fish were dried to constant weight at 80 ◦C. The dry weight of the individuals was calculated. The growth rate was expressed in terms of mg dry substance gained g−1 live fish−1 day−1

- Parameters analysed / observed: see above

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

other: Oreochromis mossambicus

Details on test organisms:

TEST ORGANISM
- Common name: Mozambique Tilapia; African mouthbrooder, Tilapia
- Source: collected from the local pond
- Age at study initiation (mean and range, SD): Juveniles
- Weight at study initiation (mean and range, SD): 3±1 g

FEEDING DURING TEST
- Food type: fresh goat liver pieces once a day for an hour (10–11 a.m.).
- Amount: ad libitum
- Frequency: once a day for an hour (10–11 a.m.)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Hardness:

3.94 mg/L (as Ca2+ Mg2+)

Test temperature:

28°C

pH:

7.2

Dissolved oxygen:

5.6 mg/L

Nominal and measured concentrations:

Control, 10000, 14000, 18000 and 22000 mg urea/L

Details on test conditions:

TEST SYSTEM
- Test vessel: plastic trough (33 cm in diameter, 20 cm depth)
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


OTHER TEST CONDITIONS
- Photoperiod: normal light.
- Light intensity: normal light.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Growth rate on day 28

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

7 247 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: extrapolated from reported growth rates

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

14 307 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: calculated from reported growth rates

Details on results:

- Tank-average or pseudo-specific growth rates for periods 0-28 : 3.4, 2.6, 1.7, 1.016 and 0.8 mg dw/fish/day in the control, 10000, 14000, 18000 and 22000 mg urea/L treatments. The growth rate was expressed in terms of mg dry substance gained g−1 live fish−1 day−1.

The growth rate of fishes reared in urea media gradually decreased. The change in growth rate of O. mossambiucs was 3.4±0.7.2, 2.641±0.409, 1.707±0.327, 1.016±0.233 and 0.834±0.182 mg g live fish−1 d−1, in the control, 10000, 14000, 18000 and 22000 mg urea/L treatments, respectively.

- Type of and number with morphological abnormalities: none reported
- Type of and number with behavioural abnormalities: none reported
- Other biological observations: none reported
- Effect concentrations exceeding solubility of substance in test medium: no
- Incidents in the course of the test which might have influenced the results: no

Reported statistics and error estimates:

The EC10 was calculated using the reported growth rates. The relative growth rates when compared to the control were calculated (growth rate x/ growth rate control) and the % inhibition was calculated. The probit values of the % inhibition was plotted against the log concentration. The resulting linear fit had an R2 of 0.9835. The linear regression was used to calculate the EC10, EC20 and EC50.

Validity of the test:

The OECD 215 is written for juvenile rainbow trout (Oncorhynchus mykiss) and hence, the validity critera apply strictly only for that species. In the present study, anopther species was tested and hence the validity criteria cannot directly applied.

In the following, the validity criteria are analysed for their relevance for the described test with O. mossambicus:

• no control mortality reported;
• the second validity criterion describes that the trout must have grown for at least 50% within 28 days to permit the detection of the minimum variation of growth rate considered as significant. The statistical analysis of the test with O. mossambicus indicate that the lowest concentration (10000 mg urea /L) with an growth inhibition of 23 % was statistically different to the control. This demonstrates that the test system was sensitive. Moreover, the statistical evaluation for O. mossambicus was not performed on the NOEC but on the more robust ECx.
• The ASV was higher than 60%
• the water temperature was reported to be within +/-1 °C

Hence, even when a different species was used, it can be concluded that the general ideas of the validation coriteria were met

.

Validity criteria fulfilled:

not applicable

Remarks:

see "Other information and results"

Conclusions:

EC10 Oreochromis mossambicus: 7247 mg urea/L
EC50 Oreochromis mossambicus: 14307 mg urea/L

Executive summary:

In the study from Palanivelu et al. 2005, the growth rate of the fresh water fish Oreochromis mossambicus in the presence of urea was determined during 28 day semi-static exposure. Five fish per concentration were exposed. In other aspects, the test design was similar to the OECD 215.

The change in growth rate of O. mossambiucs was 3.4±0.7.2,  2.641±0.409, 1.707±0.327, 1.016±0.233 and 0.834±0.182 mg g live fish−1 d−1, in the control, 10000, 14000, 18000 and 22000 mg urea/L treatments, respectively. The growth rate of fishes reared in urea media gradually decreased and was statistically different to the control for all treated test solutions.

Based on these data, the EC10 was extrapolated to be 7247 mg/L. Based on the good fit of the linear regression (log concentration vs probit inhibition, R2=0.9835 ), the calculated EC10 is considered to be robust and reliable with restrictions for the risk assessment.

Description of key information

EC10 Oreochromis mossambicus: 7247 mg urea/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

EC10

Effect concentration:

7 247 mg/L

Additional information

In the most relevant long-term fish study (Palanivelu et al. 2005), the growth rate of the fresh water fish Oreochromis mossambicus in the presence of urea was determined during 28 day semi-static exposure. Five fish per concentration were exposed. In other aspects, the test design was similar to the OECD 215.

The change in growth rate of O. mossambiucs was 3.4±0.7.2,  2.641±0.409, 1.707±0.327, 1.016±0.233 and 0.834±0.182 mg g live fish−1 d−1, in the control, 10000, 14000, 18000 and 22000 mg urea/L treatments, respectively. The growth rate of fishes reared in urea media gradually decreased and was statistically different to the control for all treated test solutions.

Based on these data, the EC10 was extrapolated to be 7247 mg/L. Based on the good fit of the linear regression (log concentration vs probit inhibition, R2=0.9835 ), the calculated EC10 is considered to be robust and reliable with restrictions for the risk assessment.

The result demonstrates that urea is of low long-term toxicity to fish. Even if the test design which was similar to OECD 215 does not include the exposure of embryos, there is sufficient information available to conclude that an ELS test according to OECD 210 is not needed. As demonstrated by the acute FET test, zebra fish embryos are not specifically sensitive to urea. Furthermore, there is information, that urea is the predominant substance for N-excretion by fish embryos to avoid ammonia toxicity (e.g. Zimmer et al. 2017, Pilley and Wright 2000; see section "Biotransformation and kinetics"). In juvenile and adult fish, about 10 to 20 % of the nitrogen is excreted as urea (Zimmer et al. 2017). Hence, urea is a endogenously produceed substance in fish.