1,3,5-triazine-2,4,6-triamine phosphate

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

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

A semi-static test of the subacute effects of melamine to Rainbow trout (Salmo gairdneri) was conducted over a 28 day period.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The melamine stock solution was prepared in Milwaukee tap water. 54 grams of melamine was dissolved in 18 liters of water. To dissolve all of the melamine it was necessary to leave the stock solution of melamine on a magnetic stirrer for 20 hours. To achieve the desired test concentrations the melamine stock solution was combined with diluent water.
This procedure was used for every water change during the experiment.
- Evidence of undissolved material: Once during this test precipitate was seen in the high test substance concentration (3000 ppm), it was not determined whether or not the precipitate was melamine.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Rainbow trout fingerlings were obtained from Hideaway Springs Trout Farm in west Bend, Wisconsin on February 16, 1984. The fish were well acclimated to laboratory conditions before their use and held under a constant 12 hour photoperiod. The fish were randomly assigned to test concentration or control groups.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

No.

Hardness:

No information available.

Test temperature:

Control: mean 14.3 °C, range 10.6 -16°C, SD 1.1
Test group 750 ppm: mean 14.3 °C, range 10.7 -16°C, SD 1.1
Test group 1500 ppm: mean 14.4 °C, range 10.7 -16°C, SD 1.1
Test group 3000 ppm: mean 14.4 °C, range 10.8 -16°C, SD 1.1

pH:

Control: mean 7.69, range 7.71 - 7.96, SD 0.17
Test group 750ppm: mean 7.71, range 7.42 - 8.02, SD 0.15
Test group 1500ppm: mean 7.65, range 7.24 - 7.93, SD 0.17
Test group 3000ppm: mean 7.45, range 6.28 - 7.90, SD 0.49
Dilutant water:
mean: 7.26, 1 S.D.: 0.34, range: 6.90-7.84
Melamine stock:
mean: 7.34, 1 S.D.: 0.71, range: 6.38-7.94

Dissolved oxygen:

D.O. (ppm)
Control: mean 9.6, range 8.6 -10.2, SD 0.6
Test group 750ppm: mean 9.5, range 8.6 - 10.2, SD 0.6
Test group 1500ppm: mean 9.6, range 8.6 - 10.4, SD 0.8
Test group 3000ppm: mean 9.7, range 8.6 - 10.2, SD 0.6
Dilutant water:
mean: 10.4, S.D.: 0.29, range: 10.0-10.7
Melamine stock:
mean: 9.65, S.D.: 0.47, range: 9.0-10.3

Salinity:

Not applicable.

Conductivity:

No data

Nominal and measured concentrations:

Nominal concentration:
Control: 0 ppm
Test group: 750 ppm
Test group: 1500 ppm
Test group: 3000 ppm

Details on test conditions:

TEST SYSTEM
5 gallon all glass aquaria were used for this test. Each tank was filled to 10 liters with dechlorinated Milwaukee tap water. The aquaria was placed in a flow through fiberglass trough for temperature control. The tanks were labeled, covered and aerated.
- Test vessel:
- Type: covered
- Glass aquaria, size: 18.9 liters, fill volume: 10 liters
- Aeration: yes,
- Semistatic test with test media exchange once at 48h and then every fifth day thereafter
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated Milwaukee tap water
- Intervals of water quality measurement: once at 48h and then every fifth day thereafter

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

> 3 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

A semi-static test of the acute and subacute effects of melamine to Rainbow trout (Salmo gairdneri) was conducted over a 28 day period. The test concentrations were: 3000 ppm, 1500 ppm, 750 ppm and zero ppm for control. The length to weight relationships, condition factors and growth rates were computed and analyzed using data collected at the start of this test and at its termination.
The results show that the growth in length is not affected by melamine while growth in weight is affected.
The statistical no-effect level based on mortality and sublethal effects is 1500 ppm of melamine.
The estimated 28 day LC50 is >3000ppm of melamine. All observed mortalities occurred in the 3000 ppm test group. The first mortality occurred on day 10 into the test, the second on day 18 and the final mortality on day 22 into the assay.

The results of the rank sum test show that the growth in length is not affected by melamine while growth in weight is affected. The statistical no-effect level based on mortality and sublethal effects is 1500ppm of melamine. As there was no lethal response greater than 30% the analysis of the lethal response is approximate. The observed 28 day LC50 is >3000 ppm of melamine.

There was at no time during this test that a mortality rate greater than 30% was observed. All observed mortalities occurred in the 3000 ppm test group. The first mortality occurred on day 10 into the test, the second on day 18 and the final mortality on day 22 into the assay. A true estimate of the 28 day LC50 value can not be computed due to the lack of a response greater than 50%. The 28 day LC50 is then greater than 3000 ppm. Under the experimental conditions of this bioassay it appears that 3000ppm is close or at the saturation point of melamine. Once during this test precipitate was seen in the high test concentration (3000 ppm), it was not determined whether or not the precipitate was in fact melamine.

The water parameters experienced during the bioassay were within safe limits. Lengths and wet weights of the surviving test fish were measured at the termination of the test. A condition (K) factor was computed for each test fish as well as instantaneous growth rates of test groups based on mean wet weights. A log-log regression analysis of the length to weight relationship was performed.

There was no significant difference of length, weight or condition factor between the test groups of fish prior to the melamine exposure. At the termination of the bioassay the regression lines produced by each test group (tank) were compared to the original (day 0) lines. The test used for this was a multivariate regression analysis and is meant to detect the differences in slope.

All the slopes for each test group gained some steepness by the end of the exposure, but only in the control and 750 ppm groups was this increase significant. The conclusion that can be drawn from this analysis is that the growth relationship of length compared to weight has changed in the control and 750 ppm test groups while in the medium and high dose groups the relationship has not changed. In all test groups the mean K factor decreased, to say that length was increasing at a faster rate than weight. In the 1500 ppm and 3000 ppm groups there was, in fact, weight loss.

Description of key information

No data with the target substance were available. A weight of evidence approach with the read across source substance melamine (CAS 108-78-1) is applied. The studies of Adema, 1982 (DSM) and Goodrich, 1984 (American Cyanamid) with comparable reliability and adequacy were used in a weight of evidence approach. The NOEC for the egg/larvae stage was determined to be >1000 mg/L. Both results indicate a low subchronic toxicity to developmental fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

1 500 mg/L

Additional information

Scientifically it is not regarded necessary to conduct long-term toxicity studies in fish. In the acute toxicity studies it could be shown that aquatic invertebrates are more sensitive compared to fish. Therefore, a chronic toxicity study to Daphnia magna was conducted. This study did not reveal any toxic effect up to a concentration of 10 mg/L. Therfefore, a NOEC of 10 mg/L was determined. Nevertheless, some long-term toxicity data for fish exist and need to be mentioned.

 

No data with the test substance were available. A weight of evidence approach with the read across substance melamine (CAS 108-78-1) is applied. Please refer section 13 for the read across justification. As the substance is a salt of phosphate, effects can be read-across from the cation, which is likely to have the more important effects. In biological systems, phosphate is an abundant in cells and body fluids. In ecological terms, because of its important role in biological systems, phosphate is a highly sought-after resource. Thus, read-across from component CAS Reg.-No. 108-78-1, melamine, is justified. All effect values are minimum values as the molar ratio anion:cation equals 1.74.

 

Three investigations with 2 species of fresh water fish are available. A study on the egg/larvae development of Jordanella floridae (Adema, 1982 (DSM), a study with juvenile rainbow trout (Goodrich, 1984 (American Cyanamid) and an egg/larvae developmental study with rainbow trout (Ramusino 1982). The studies of Adema, 1982 (DSM) and Goodrich, 1984 (American Cyanamid) with ca. the same reliability and adequacy were used in a weight of evidence approach. 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. This is interpreted by the authors as a "generic effect". No bias control measures were described, which increases the suspicion that an artefact is reported. This study is therefore not included in the weight of evidence approach. Even if the result would not be an artefact, it would not cause a classification of melamine, as effects occurred above 100 mg/L.

 

In conclusion, in light of the chronic toxicity study to Daphnia magna and the available fish studies on different species an additional chronic toxicity study to fish is scientifically and for reasons of animal welfare not justified