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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented study report, with a method similar to present guidelines.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Semi-static test of acute and subacute effects to Rainbow trout over 28 days.
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 (3000ppm), 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 informaion available.

Test temperature:
Control: mean 14.3 °C, range 10.6 -16°C, SD 1.1
Test group 750ppm: mean 14.3 °C, range 10.7 -16°C, SD 1.1
Test group 1500ppm: mean 14.4 °C, range 10.7 -16°C, SD 1.1
Test group 3000ppm: 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, S.D.: 0.34, range: 6.90-7.84
Melamine stock:
mean: 7.34, 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, 1 S.D.: 0.29, range: 10.0-10.7
Melamine stock:
mean: 9.65, 1 S.D.: 0.47, range: 9.0-10.3
Salinity:
Not applicable.

Nominal and measured concentrations:
Nominal concentration:
Control: 0ppm
Test group: 750ppm
Test group: 1500ppm
Test group: 3000ppm

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:
96 h
Dose descriptor:
LC50
Effect conc.:
> 3 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
3 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
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 3000 ppm is close or at the saturation point of melamine. Once during this test precipitate was seen in the high test concentration (3000ppm), 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 750ppm 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 750ppm 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 1500ppm and 3000ppm groups there was, in fact, weight loss.

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.
Executive summary:

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 short term toxicity to fish endpoints are:

NOEC 96h: 3000 mg/L

LC50 96h: 3000 mg/L

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Secondary source with very limited data given.
Guideline:
other: no data
Principles of method if other than guideline:
96 h acute toxicity test with guppies
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Analytical monitoring:
not specified
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
saturated melamine solution in water (4.4 g/L)
Test organisms (species):
Poecilia reticulata
Details on test organisms:
no data
Test type:
not specified
Water media type:
not specified
Total exposure duration:
96 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
saturated melamine solution in water (4.4 g/L)
Details on test conditions:
no data
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
LC10
Effect conc.:
> 4 400 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 4 400 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data
Sublethal observations / clinical signs:

After 96 hours exposure to a saturated melamine solution in water (4.4 g/L) the mortality was less than 10%, and it appeared to be impossible to establish an LC50.

Executive summary:

An acute toxicity test with guppies was performed. After 96 hours exposure to a saturated melamine solution in water (4.4 g/L) the mortality was less than 10%, and it appeared to be impossible to establish an LC50.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Database source, only few details are provided, but data base is created and used by the Japanese authorities, and is therefore considered to be sufficiently reliable.
Guideline:
other: not stated
Principles of method if other than guideline:
Fish toxicity test.
GLP compliance:
not specified
Remarks:
no information on GLP compliance available
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Analytical monitoring:
not specified
Details on sampling:
no data
Vehicle:
not specified
Details on test solutions:
no data
Test organisms (species):
Oryzias latipes
Details on test organisms:
no data
Test type:
semi-static
Water media type:
not specified
Total exposure duration:
48 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
no data
pH:
no data
Dissolved oxygen:
no data
Salinity:
no data
Nominal and measured concentrations:
no data
Details on test conditions:
no data
Reference substance (positive control):
not specified
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
mortality (fish)
Details on results:
no data
Results with reference substance (positive control):
no data
Reported statistics and error estimates:
no data
Executive summary:

The LC50,48h (Oryzias latipes) is >1000 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1979
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Very short report with only very few details reported.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Fish toxicity test.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Leuciscus idus
Details on test organisms:
Length: 5.78 cm.
Mass: 2.50 g.
3 fish per group.
Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No data.
Hardness:
No data.
Test temperature:
No data.
pH:
7
Dissolved oxygen:
No data.
Salinity:
No data.
Nominal and measured concentrations:
Pretest: 100 - 500 - 1000 - 3000 - 5000 - 10000 mg/L
Details on test conditions:
No data.
Reference substance (positive control):
yes
Remarks:
KCN: LC50 = 0.74 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 500 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
mortality (fish)
Details on results:
No data.
Results with reference substance (positive control):
No data.
Reported statistics and error estimates:
No data.
Executive summary:

LC50 48h > 500 mg/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Very short report without much details.
Qualifier:
according to guideline
Guideline:
other: standard NEN 6504
Principles of method if other than guideline:
Standard NEN 6504
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Test organisms (species):
Poecilia reticulata
Test type:
static
Water media type:
not specified
Limit test:
no
Total exposure duration:
96 h
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 4.59 g/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Sublethal observations / clinical signs:

After 96 hours 3 fish in the two lower concentrations and 4 fish in the highest concentration had died.

Executive summary:

A fish acute toxicity test for Melamine was conducted with Poecilia reticulata. After 96 hours of exposure to 0, 1.14, 2.31, and 4.59 g/L Melamine a mortality rate of 20 % was observed in the highest test substance group.

Therefore the LC50 value is above 4.59 g/L.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, data originate in part from a secondary source
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 100 mg/L. Samples were taken after 1, 24, 48, 72, and 96 hours.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Tap water was passed through a membrane filter (1 μm) to remove particulate matter and then a high-grade activated carbon filter to remove any organic contaminants. Preliminary solubility trials indicated that this test substance did not produce test solution of 100 mg/L using organic solvents such as acetone, dimethyl sulfoxide (DMSO), ethanol, and N,N-dimethylformamide (DMF). Therefore, the test solution was prepared by direct addition of the test substance to dilution water.
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Japanese rice fish
Source: Korea Institute of Toxicology (KIT), KRICT, Deajeon, Korea
- Age at study initiation (mean and range, SD): 3-4 month
- Length at study initiation (length definition, mean, range and SD): 2.0±1.0 cm
- Weight at study initiation (mean and range, SD): 0.19 g (S.D. = 0.03 g)
- Feeding during test: none


ACCLIMATION
- Acclimation period: 7 days
- Acclimation conditions: same as in test
- Feeding frequency: not fed 24 hours prior to test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
10-250 mg/L CaCO3
Test temperature:
23.3 ± 0.1 °C
pH:
7.18-7.97
Dissolved oxygen:
5.2-8.5 mg/L
Salinity:
<0.01 mg/L (residual chlorine)
Nominal and measured concentrations:
Nominal test substance concentration: 100 mg/L
The concentrations of the test substance were measured to be 13.0-14.2 mg/L at a nominal concentration of 100 mg/L (13.0-14.2% of nominal concentration) at 0, 48, and 96 hours.
Details on test conditions:
TEST SYSTEM
- Aeration:none
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Filtered tap water (1 µm, membrane filter), residual chlorine or organic substances were removed by filtering through an activated carbon filter.
- Chlorine: < 0.01 mg/L


OTHER TEST CONDITIONS
- Photoperiod: 16:8 h day-night regime
- Light intensity: 990-1060 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality and abnormality after 1, 24, 48, 72, and 96 hours

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.1, 1, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: no mortality observed at 100 mg/L
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 13.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
> 13.5 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
There were no mortalities and adverse effects observed in any of the fish exposed to control and 13.5 mg/L treatment effect group during the test. All results were expressed in terms of the mean measured concentration.
Reported statistics and error estimates:
No statistics performed
Sublethal observations / clinical signs:

Table 1. Cumulative mortality of Oryzias latipes

Nominal concentration (mg/L)

Mean measured concentration (mg/L)

Number of organisms tested

Cumulative number of dead fish

24-hour

48-hour

72-hour

96-hour

Control

ND

7

0

0

0

0

100

13.5

7

0

0

0

0

* ND : Not detected

Table 2. Symptoms of intoxication of the test substance to Oryzias latipes

Nominal concentration (mg/L)

Mean measured concentration (mg/L)

Symptoms of Intoxication

24-hour

48-hour

72-hour

96-hour

Control

ND

NOR(7)

NOR(7)

NOR(7)

NOR(7)

100

13.5

NOR(7)

NOR(7)

NOR(7)

NOR(7)

* ND : Not detected

* ( ) : Number of fish

* NOR : Normal

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to the analogue justification attached to section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
mortality (fish)
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Nite 2010
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 3 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Goodrich & Lech 1984.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Source, WoE, RA-A, CAS 7757-93-9, Kim et al. 2013
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 500 mg/L
Nominal / measured:
not specified
Conc. based on:
not specified
Basis for effect:
mortality (fish)
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Pagga 1979
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 4 400 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Jansen 1978
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 4.59 g/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Source, WoE, RA-A, CAS 108-78-1, Vaessen 1982

Description of key information

 No toxic effects on fish were recorded within the limit of water solubility of the substance.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

Data on the acute toxicity of the target substance diphosphoric acid, compound with 1,3,5-triamine (1:2) to fish are not available. The evaluation of the acute toxicity to fish is based on a weight of evidence approach studies conducted with available data on the source substances melamine (CAS 108-78-7) and calcium hydrogenorthophosphate (CAS 7757-93-9). Whereby melamine represents the 1,3,5-triamine moiety of the target substance. The source substance calcium hydrogenorthophosphate was selected to assess the toxicity of the pyrophosphate moiety. The read across approach is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Further justification is given in the analogue justification in IUCLID section 13.

Two reliable studies on the acute toxicity of melamine (CAS 108-78-1) are available. One of the studies investigated the acute toxicity of the substance to Oryzias latipes in a semi-static test system. After an exposure period of 48 hours a LC50 (48 h) >1000 mg/L (nominal) was determined (NITE, 2010). The second study investigated the acute and sub-acute effects on rainbow trout (Oncorhynchus mykiss) in a semi-static test. The fish were exposed to test substance concentrations of 750, 1500 and 3000 ppm over a total exposure period of 28 days. The test media was exchanged once at 48 h and then every fifth day thereafter. Mortality or behavioural changes were recorded every 24 hours. The determined LC50 (96 h) was > 3000 mg/L (nominal) (Goodrich & Lech, 1984).
The results are supported by three further tests rated with a low reliability (4) due to the limited available documentation. The determined LC50 values were: LC50 (48 h) >500 mg/L for Leuciscus idus (Pagga, 1979) and LC50 (96 h) >4400 mg/L for Poecilia reticulate (Jansen, 1978; Vaessen, 1982) respectively.

The acute toxicity of the source substance calcium hydrogenorthophosphate (CAS 7757-93-9)to fish was investigated in a study following OECD guideline 203 (Kim et al. 2013). The test was set up as a limit test under static conditions. Fish of the species Oryzias latipes were exposed to a nominal test substance concentration of 100 mg/L. The analytical recovery rate of the test substance was 13.0-14.2% of the nominal concentration. During the test period of 96 hours mortality and abnormal behaviour of the test organisms were examined after 1, 24, 48, 72, and 96 hours. Mortality or adverse effects of the test substance were not observed. Based on the mean measured concentration a LC50 (96 h) > 13.5 mg/L (nominal: LC50 (96 h) >100 mg/L) was determined.

Based on the available data for the source substances the target substance diphosphoric acid, compound with 1,3,5-triamine (1:2) is considered not acutely toxic to fish.