Melamine

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 72-4 (Fish Early Life-Stage and Aquatic Invertebrate Life-Cycle Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Concentration control samples were further collected from an alternating replicate per test group on days 0, 8, 15, 22, 29 and 36 (generally after replacing the stock solutions) to determine concentrations of the test substance in the test vessels.
Samples from all test groups were taken from the middle of the test vessel using a glass pipette and sealed in glass ampoules. The samples were transferred to the analytical laboratory and analysed generally directly after sampling. If this was not possible, the samples were stored in a freezer until analysis.

Vehicle:

yes

Details on test solutions:

The test water (dilution water) was aerated non chlorinated drinking water obtained from the municipal water works of the city of Frankenthal (67227, Germany), additionally purified through a charcoal filter and diluted with deionized water to achieve a hardness of approximately 100 mg/L CaCO3. The mixed test water was sanitized by UV treatment prior to entering the aquaria. Generally the total organic carbon (TOC) content of the dilution water is <2.0 mg/L.
A stock solution of the test substance in dilution water (500 mg/L) was prepared at least weekly. For preparation, 35 g of the test substance was weighed into a beaker and mixed with an aliquot of dilution water from the stock solution tank (70 L) using an ultra turrax high shear mixer. The mixture was then poured into the stock solution tank and the whole test batch was homogenized again with an ultra turrax. The stock solution was further mixed with an overhead winged stirrer for approximately one hour. Prior to further use, the stock solution was checked visually to ensure that all test substance was completely dissolved.
Throughout the exposure period a metering pump delivered the stock solution to each mixing tank where it was continuously diluted with aerated dilution water to generate the nominal test concentration for each test group. The concentration of the solution in the stock solution tank was verified by analysis (without a GLP-status) after each fresh preparation before use. The results were used as the basis to adjust the rate of the stock solution pumps as needed.
The stock solution was diluted to obtain the target nominal concentrations.

Test organisms (species):

Pimephales promelas

Details on test organisms:

The parental fathead minnows were supplied by Osage Catfisheries, Inc. (Osage Beach, MO 65065, USA). Parent fish were acclimatized to testing conditions including test water (dilution water), temperature, and photoperiod for at least 14 days prior to egg collection. The parent fish were observed to be in good health and the mortality was below 5% in the last 2 weeks before the test was started.
Acclimatized parent fish were kept in flow-through glass aquaria (45 L) divided into three spawning groups consisting of either 1 male and 2 females or 2 males and 3 females. Mature fish were sexed according to external secondary sexual characteristics. A total of 18 spawning groups (6 aquaria) were used to produce eggs to initiate the exposure. Each spawning group was provided with spawning tiles (a stainless steel half pipe) at least equivalent to the number of males. On day 0 of exposure, fish were allowed to spawn from 09:10 am to 10:30 am and afterwards egg clutches were collected from 7 spawning tiles. The eggs were carefully removed from the tile, washed several times and were pooled. Subsequently, the necessary number of eggs was transferred into the test vessels. This was finished by 12:50 pm. Thus it is ensured that eggs used in the study were fertilized less than 4 hours before the start of the exposure. The developmental stage at test initiation was confirmed by examination of a representative sample of eggs using a stereo microscope after the fertilized eggs were impartially transferred to the test vessels (13:00 pm). The embryos were in the blastula developmental phase at approx. the 1000 cell stage.
Each test group initially consists of 100 fertilized eggs and 4 replicates (25 fertilized eggs/replicate).

Feeding was initiated in all test groups at the end of swim up (day 6) with live brine shrimp nauplii (Artemia sp.) and a fine milled commercial fish diet (Tetramin Tetra-Werke, Melle, Germany). Feeding was conducted at least twice daily on workdays and once or twice daily on non-working days. The combination of live Artemia and commercial food was fed in increasing quantity during the test corresponding to the size of the fish and was continued until one day before the termination of exposure.

Test type:

flow-through

Water media type:

freshwater

Total exposure duration:

36

Remarks on exposure duration:

d

Post exposure observation period:

None.

Hardness:

100 mg CaCO3/L.

Test temperature:

24.5 - 25.4 °C.

pH:

7.8 - 8.1

Dissolved oxygen:

5.8 - 8.5 mg/L.
Oxygen values decreased to 69 % of the saturation value (8.38 mg/L at 25 °C) on Day 28. Therefore aeration was started on Day28in all vessels.

Salinity:

/

Nominal and measured concentrations:

Nominal concentrations: 0, 0.625, 1.25, 2.5, 5.0 and 10 mg/L
Mean measured concentrations:

Details on test conditions:

The test was conducted in a continuous flow through system. Tempered (25 ±1 °C) dilution water flowed into a separate mixing tank for each test group at a rate controlled by rotameters. Metering pumps were used to proportionally deliver the test substance stock solution into the mixing tank for each exposed test group where it was instantly diluted to obtain the desired test concentration. From the mixing tank, test solution flows through a flow splitter splitting the test solution evenly by four glass tubes of the same inner diameter and length, each of which lead into one of the four replicate test vessels per test group.
Fertilized eggs (embryos) and larvae were exposed in cylindrical glass vessels with a diameter of 19 cm and a screened outlet at a height of 6 cm which maintained a constant water volume of approximately 1.7 litres. The test solution was introduced into this test vessel above the water surface. The outlet empties into a larger stainless steel aquarium, into which the juveniles were transferred at a suitable size for the remainder of exposure. These aquaria (inner dimensions: 29 cm long, 21 cm wide, 22 cm high) contained 9 L of test solution maintained by an overflow located 15 cm above the bottom covered with a stainless steel screen.
Generally aquaria were not aerated. However in response to a low dissolved oxygen measurement on study day 28 (5.8 mg/L, approx. 69% of saturation), slight aeration via glass tubes was provided in all test replicates through the end of exposure. The temperature in the exposure room was regulated at 25°C and the aquaria were provided dim light (about 101–209 Lux under the lid) at an automatically maintained photoperiod of 16 hours light and 8 hours darkness.
The construction materials used for the exposure apparatus did not contain any significant amounts of leachable substances nor are they expected to bind any substantial amounts of the test substance. Plastics were used only where absolutely necessary. Most of the apparatus consisted of glass or stainless steel. The tubes through which the stock solution was metered into the mixing tanks were made of Teflon.

Each test group initially consists of 100 fertilized eggs and 4 replicates (25 fertilized eggs/replicate).

Reference substance (positive control):

not specified

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

>= 5.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

length

Duration:

36 d

Dose descriptor:

NOEC

Effect conc.:

>= 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

36 d

Dose descriptor:

LOEC

Effect conc.:

10.1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

length

Details on results:

All individually measured concentrations during the exposure period were within the range ±20% of the overall mean measured concentrations for each treatment group.
For biological results see also the attached Tables in the file 'Results of the fish early life-stage tox study.docx'.
The most sensitive endpoint was growth (total length).
There was no statistically significant decrease in hatching success (i.e. embryo survival until end of hatch) in any of the treatment groups in comparison to the control group when data were evaluated using Jonkheere-Terpstra test (one-sided) and the Wilcoxon test (one-sided).
There was no statistically significant decrease in survival from hatch until the end of swim up in any of the treatment groups in comparison to the control group when data were evaluated using Jonkheere-Terpstra test (one-sided) and the Wilcoxon test (one-sided).
There was a statistically significant decrease in fish survival from end of swim-up (day 6) until the end of exposure (day 36) in the test group 5 (10.0 mg/L) in comparison to the control group using Jonkheere-Terpstra test (one-sided) (*p ≤0.05).
Overall survival was statistically significantly decreased in the treatment group 5 (10.0 mg/L) in comparison to the control group using Jonkheere-Terpstra test (one-sided) (*p ≤0.05).
No substance-related effect was observed on time to hatch, hatching success, time to swim-up and larval survival to swim-up. Juvenile survival (day 6–36) as well as the overall survival (day 0–36) was statistically significantly decreased in the treatment groups 10.0 mg/L (nominal concentration) in comparison to the control group. Mortality occurred predominantly during the juvenile period after swim-up. No test substance-related effect on survival was seen in the test concentrations ≤5.0 mg/L (nominal).

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

See above under 'Details on results'.

Signs of toxicity and abnormalities:

No abnormalities were seen in the treatment groups 0.625, 1.25, 2.5 and 5.0 mg/L (nominal concentration) and in the control group. In the treatment group 5, deformations in two fish were recorded from day 31 to the end of exposure. It is unclear if these were spontaneous findings or an effect of the test substance, since the effect on survival and growth endpoints was very minor. Due to the low incidence these finding are not necessarily an indication for a specific mode of action.

 

Growth:

In comparison to the control group the mean wet weights of the surviving fish in all test groups at the end of the exposure period were not statistically significantly decreased or increased. However, for the parameter length a slight decrease of -5.9% in comparison to the control group was statistically significant in the test group 5 (10.0 mg/L). Despite the decrease in body length, mean wet weight in test group 5 was 4.3% more than the control. Although the decrease in body length did not correspond to any decrease in body weight, the slight effect on survival and presence of 2 abnormal individuals in this test group indicate an overall biological effect.

 

Water quality data:

The instantaneous test temperature was generally 25 ±1°C in all aquaria throughout the exposure period. Continuous temperature measurement in one test vessel of the control group ranged from 24.5–25.4°C. Over the 36 day exposure period, the mean temperature in the control vessel was 25.1 °C.

The pH was nearly constant during the whole exposure period and was in a range of 7.8–8.1. The concentrations of dissolved oxygen were maintained in a range between 5.8 and 8.5 mg/L corresponding to approximately 69–101% of the maximum saturation at the test temperature of 25 °C. On day 28 the dissolved oxygen concentration was 5.8 mg/L (69% of the saturation concentration) in test group 1, replicate B and from then on slight aeration was administered to ensure sufficient oxygen content. The measured values for hardness were 1.05–1.09 mmol/L (approx. 105–109 mg/L CaCO3) at study day 0 and 1.07–1.18 mmol/L (approx. 107–118 mg/L CaCO3) at the end of exposure.

Consequently, measurements of pH, dissolved oxygen, and hardness were comparable between the control and treatment groups and did not appear to be influenced by test substance concentration.

 

Validity criteria fulfilled:

yes

Conclusions:

NOEC >=5.1 mg/L based on mean measured concentrations.

Executive summary:

The 36-day chronic toxicity of melamine to early life stages of fathead minnow (Pimephales promelas) was studied under flow-through conditions following the OECD 210 and the EPA-OPP guidelines. Fertilized eggs (<4 hours old) were obtained from 7 clutches, and 100 embryos evenly distributed among four replicates per test group. The test groups comprised 5 concentrations of the test substance and a dilution water control: 0 (Control), 0.625, 1.25, 2.5, 5.0 and 10 mg/L as nominal concentrations, corresponding to mean measured concentrations of <wLoQ., 0.618, 1.18, 2.30, 5.25 and 10.1 mg/L. The exposure system was maintained with dissolved oxygen levels >60% saturation at 25 °C and a pH of 7.8 to 8.1.

The following effects were observed in the test groups, nominal concentration (mean measured):

Control: Conformed to all quality and validity criteria

0.625 mg/L: No test substance-related effects

1.25 mg/L: No test substance-related effects

2.5 mg/L: No test substance-related effects

5.0 mg/L: No test substance-related effects

10.0 mg/L: Slightly decreased survival rates after swim up, 2 deformed individuals and slightly reduced growth (as body length) after swim up in the surviving individuals

 

The individually measured concentrations during the exposure period were within the range ±20% of the overall mean measured concentrations. The mean measured concentrations are considered an accurate representation of the actual exposure concentrations over the course of the test.

Under the conditions of this study the most sensitive endpoint was growth as total length. The overall no observed effect concentration (NOEC) was 5.1 mg/L based on mean measured concentrations and 5.0 mg/L as the nominal concentration. The overall lowest observed effect concentration (LOEC) was 10.1 mg/L based on mean measured concentrations and 10.0 mg/L as the nominal concentration.

The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. All water quality and environmental parameters monitored were within the required ranges. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Description of key information

Melamine is of low long-term toxicity to fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

5.1 mg/L

Additional information

The key study of Salinas 2015 was performed with fathead minnow according to the OECD method 211 with GLP. The NOEC was >=5.1 mg/L, based on slightly reduced body length, slightly decreased survival rate and 2 deformed individuals at 10.1 mg/L.

 

Four further long-term investigations are available. A study on the egg/larvae development of Jordanella floridae (Adema 1982), a study with juvenile rainbow trout (Goodrich 1984) and an egg/larvae developmental study with rainbow trout (Ramusino 1982). The studies of Adema 1982 and Goodrich 1984 have ca. the same reliability and adequacy. The NOEC for the egg/larvae stage is >1000 mg/L, the NOEC of the juvenile fish for 28 d exposure is 1500 mg/L. Both results indicate a low subchronic toxicity to fish.

The study of Ramusino 1982 is considered to be not sufficiently reliable. The NOEC based on mortality is 1000 mg/L. Doubtful histological results, as judged e.g. by the missing dose-response relation, with 125 mg/L producing more, only histologically detected malformed embryos than at 250 or 500 mg/L.

The fourth study is a 8-weeks feeding study with sex-reversed red tilapia by Phromkunthong et al., 2013. The feeding trial (inclusion levels: 5 - 30 g/kg feed) investigated growth performance, feed efficiency, histopathological changes and melamine residues. Fish grew less, utilized feeds less efficiently and performed poorly, besides exhibiting various defects such as fin erosion, anorexia, sluggish swimming behaviour, paling/darkening of skin and low survival at the high concentrations of melamine in feed, used in this study.