1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

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:

from 27 Feb 2015 to 01 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Batch number: 17321797V0
- Expiry date: 27 September 2041

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the stock solutions were analytically determined during the study (without GLP status) and used as a basis to prepare the test solution. Aliquots of the samples were filled into into vials and then the aliquots were directly measured. Aliquots therefrom were further diluted with water (concentration range: 0.10692 mg/L to 29.52 mg/L). The overall mean measured concentration from the stock solutions was 25.4 mg/L.

- Sampling method: Analytical concentration controls in the test vessels were conducted at start of exposure (study day 0), before insertion of fertilized eggs and then once weekly. All 4 replicates per test group were sampled at the same time at the start of the test (0 day) to demonstrate equivalent exposure conditions, then weekly over one renewal interval from one alternating replicate. Samples from all test groups were collected directly from the middle of the test vessel using a glass pipette and were placed into glass sample vials.
The required volume (20 mL) of the sample was fixed in agreement with the analytical laboratory prior to the first sampling. The samples were transferred to the analytical laboratory and analyzed generally directly after sampling.
- Sample storage conditions before analysis: The samples were stored under ambient conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A WAF was prepared. A saturated stock solution was prepared using a generator system as recommended in OECD Guidance Document No. 23 according to the following procedure: A tank containing 10 L test water was connected via tubes with a filter (type Micro-Klean B2, Cuno Europe) and a pump (Hydrovar, 1.1 KW, Lowara (used for test group 1) and Multi Eco 34.6 P, KSB, used for control group). The filter was pre-washed by circulating test water through the system for about 30 minutes. The rinse water was subsequently discarded. Then 18 g of the test item was placed on the washed water-moistened cellulose filter and the system connected to a tank containing 180 L test water. The pump circulated the water at a rate of approximately 4 to 6 m3 per hour through this filter for a period of about 2 days. Afterwards, the pump was stopped and the stock solution was allowed to acclimate to test temperature for 1 day and was used then for up to 15 days. The solution for the control group was treated in the same way as the test substance groups but without test substance.
- Controls:
Blank Control: Test solution without test substance
Positive Control: Not performed

- Evidence of undissolved material: The stock solutions were visibly clear and colorless.
- Homogeneity and stability of the test solution: The stability of the test substance in aqueous solution over a period of 17 days has been demonstrated and the true dissolution of the test substance was shown by analyzing filtered samples.
The homogeneity of the test substance was demonstrated by analyzing filtered and unfiltered samples of the stock solution prior to the start of exposure. The concentration difference between filtered and un-filtered stock solution concentration was < 20%.

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnow (Pimephales promelas)
- Source: Laboratory of Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
- Age at study initiation: < 4 hours old
- Method of breeding: The parental fathead minnows (Pimephales promelas) were bred at the testing facility and all originate from the same batch of fish.
- Feeding during test: Feeding was initiated in all test groups at the end of hatch (day 6)
- Food type: Feeding was initiated with live brine shrimp nauplii (Artemia sp.) and a fine milled commercial fish diet (“Tetramin” Tetra-Werke, Melle.
Germany).
- Amount: 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.
- Frequency: Feeding was conducted at least twice daily on workdays and once or twice daily
on non-working days.

ACCLIMATION
- Acclimation period: 2 weeks
- Acclimation conditions: The fish were kept in glass tanks at 25°C, with an oxygen content > 60% saturation in the dilution water used in the test. 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.
- Type and amount of food: Live brine shrimp nauplii (Artemia sp.) and a fine milled commercial fish diet (“Tetramin” Tetra-Werke, Melle, Germany)
- Feeding frequency during acclimation: At least twice daily on workdays and once or twice daily on non-working days.
- Health during acclimation (any mortality observed): The fish were not medically treated. The parent fish were observed to be in good health and there was no mortality in the last 2 weeks before the test was started.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

33 d

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

114 – 133 mg/L CaCO3

Test temperature:

24°C

pH:

7.8 – 8.5

Dissolved oxygen:

≥ 60 mg/L

Salinity:

Not applicable

Conductivity:

255 - 256 μS/cm

Nominal and measured concentrations:

Nominal concentration: 0 (control) , 10 mg/L
Measured concentratios: < 0.05 mg/L, 10.2 mg/L (104% recovery)

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass tanks. Fertilized eggs (embryos) and larvae were exposed in cylindrical glass vessels with a constant water volume of approx. 1.5 liter. Juveniles were exposed in glass aquaria (approx. 10 L) with transparent lids.
- Fill volume: 45 Liters
- Aeration: Generally aquaria were not aerated. However in response to a low dissolved oxygen
measurement on study day 10 (5.8 mg/L, approx. 68 % of saturation), slight aeration via glass
tubes was provided in all test replicates through the end of exposure.
- Renewal rate of test solution: 3 times per week. On renewal days, the exchange of the test solutions was generally performed by removing approx. two thirds of the old test solution in the test vessels and replacing it with freshly prepared test solution to maintain the required volume, approx.1.5 L or approx. 10 L respectively. On non-renewal days, the volume of syphoned off test solutions were replaced by the required volume of fresh tests solutions after the daily cleaning procedure.
- No. of organisms per vessel: The parent fish were kept divided into three spawning groups consisting of either 1 male and 2 females or 2 males and 3 females.1 male and 2 females or 2 males and 3 females. A total of 15 spawning groups (5 aquaria) were used to produce eggs to initiate the exposure.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 3
- Biomass loading rate: The maximum biomass loading at the end of the test, based on the mean wet weight of all individual replicates for all test groups, was 0.42 g fish/L
- Hatching: Each spawning group was provided with spawning tiles (a stainless steel half pipe) at least equivalent to the number of males and the fish are allowed to spawn for several hours. On day 0 of exposure, fish were allowed to spawn from 08:30 am to 11:30 am and afterwards egg clutches were collected from 3 spawning tiles (approximately 100 eggs/tile). 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 12:10 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 (Zeiss SteREO Discovery.V12, Carl Zeiss Jena GmbH, 07745 Jena, Germany) after the fertilized eggs were impartially transferred to the test vessels (12:10 pm). The embryos were in the blastula developmental phase at approx. high stage.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 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 dilution water was sanitized by UV treatment prior to entering the aquaria. The drinking water was regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the department Environmental Analytics Water/Steam Monitoring of BASF SE as well as for presence of microbes by a contract laboratory. The drinking water was free of heavy metals and impurities and was considered to be acceptable for the purpose of this study. The German Drinking Water Regulation (Trinkwasserverordnung) served as a guideline for maximum tolerable contaminants.
- Total organic carbon: Generally the total organic carbon (TOC) content of the dilution water is <2.0 mg/L. On day 18 of the test, the total organic carbon (TOC) content in the test water of the control group was determined at 1.4 mg/L.
- Conductivity: The test water had a specific conductivity of 255 μS/cm on day 0 and 256 μS/cm on day 33.
- Acid capacity: The acid capacity [K] (concentration of the hydrochloric acid for titration 0.02 mol/L) in the test water of the control group ranged from 2.34–2.36 mmol/L at the start and end of exposure.
- Culture medium different from test medium: No
- Intervals of water quality measurement: The test temperature was measured daily in alternating replicates of each test group using an alcohol thermometer. Additionally the temperature was measured and recorded continuously in one aquarium of the control group with an EBI Winlog 2000 automatic logger (Ebro Electronic GmbH & Co. KG, Ingolstadt, Germany).
The pH of the test water was measured in the old and in the freshly prepared test solutions in alternating replicates of each concentration for one interval per week using a WTW pH/Oxi 340i combination meter (WTW GmbH, Weilheim, Germany).
Dissolved oxygen content in all replicates of each test group was measured in the old and in the freshly prepared test solutions once weekly using a WTW pH/Oxi 340i combination meter (WTW GmbH, Weilheim, Germany).
The total hardness of the test water from all test vessels was determined at start (day 0) and end of the exposure (day 33) by titration with EDTA.
The conductivity of the dilution water was measured at the start (day 0) and end of the exposure (day 33) with a WTW LF320/SET meter (WTW GmbH, Weilheim, Germany).
The acid capacity of the dilution water was determined at the start (day 0) and end of the exposure (day 33) by titration with HCl. The light intensity was measured once during the exposure period with a Testo 545 (Testo AG, Lenzkirch, Germany).
The content of total organic carbon (TOC) within the test water of the control group was determined at the Biodegradation Laboratory of the Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany in compliance with the Principles of Good Laboratory Practice.
TOC was determined using a Shimadzu TOC VCSN analyzer (Shimadzu Corp., Japan).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness.
- Light intensity: About 180–231 Lux

EFFECT PARAMETERS MEASURED The objective of the study was to assess the effects of the test substance on the time to hatch, hatching success, survival and growth of the fathead minnow (Pimephales promelas) during early life-stage development, thus establishing chronic toxicity levels for the most critical and sensitive life-stages of a freshwater fish.
The following endpoints were evaluated from these observations: time to hatch and swim up, hatching success, stage-specific and overall survival, overall growth, and sublethal, morphological or behavioral effects. Hatching success was calculated as the percentage of embryos that hatched successfully, irrespective of larval survival. Stage-specific survival was calculated from the number of embryos, larvae, or juvenile fish that survived as a percentage of the number of survivors from the preceding stage. Overall survival was calculated as the fraction of surviving fish at test termination from the number of embryos at test initiation.
From the first day of exposure until hatching was complete, eggs were observed daily for hatching and mortality.

TEST CONCENTRATIONS
- Nominal concentration: 0 (control) mg/L and Limit test
- Measured concentration (mean): < 0.1 mg/L, 10.2 mg/L (104 % recovery)

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: In a previous acute fish study the test substance did not show a toxic effect up to 10 g/L. The highest recommended test concentration for chronic studies is 10 mg/L. Since no adverse chronic effect is expected, t is test was performed as a limit test at the chronic limit concentration of 10 mg/L.

Reference substance (positive control):

no

Key result

Duration:

33 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

33 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Key result

Duration:

33 d

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

number hatched

Details on results:

- Hatching and Survival: Hatching started simultaneously in all test groups on day 2 and was complete by day 5. No test substance-related effect was observed on the time to start or end of hatching. Hatching success ranged from 92–96% for the replicates of the control group and there was no statistically significant decrease in hatching success in the treatment group in comparison to the control group. Survival from hatch to the end of swim-up (day 6) was 96–100% for the replicates of the control group and there was no statistically significant decrease in survival in any of the treatment groups in comparison to the control group. In all test groups, larval swim-up started on day 4 of exposure and was complete simultaneously with the control on day 6. No test substance-related effect was observed on the time to swim-up.
From the end of swim-up to the end of exposure (day 6–33) survival was 100% for the replicates of the control group. The survival from the end of swim-up to the end of exposure (day 6–33) as well as the overall survival (day 0–33) was not statistically significant in comparison to the control group. There was no statistically significant test substance-related effect on survival in test concentration 10 mg/L. The NOEC for survival was ≥10 mg/L (nominal concentration). The LOEC for survival was >10 mg/L (nominal concentration).
- Signs of toxicity and abnormalities: There were no signs of toxicity or abnormalities observed among the replicates of the control group and the treatment group.
- Growth: In comparison to the control group the mean wet weights and the total body lengths of the surviving fish at the end of the exposure period were not statistically significantly decreased in the treatment group. There was no test substance related effect on growth. In conclusion, under the conditions of this study exposure of Melamincyanurat prepared at a nominal concentration of 10 mg/L over 33 days resulted in no adverse effects observed on the time to hatch, hatching success, survival or growth of the fathead minnow (Pimephales promelas) during early life-stage development. The overall no observed effect concentration (NOEC) was ≥10 mg/L (nominal concentration). The overall lowest observed effect concentration (LOEC) was >10 mg/L (nominal concentration).

Results with reference substance (positive control):

No test with reference substance was performed.

Reported statistics and error estimates:

The following parameters were subject to statistical analyses:
• hatching success (survival of embryos day 0–end of hatch)
• larvae survival from end of hatch–day 6 (end of hatch until end of swim-up)
• juvenile survival from days 6–33 (end of swim-up until end of exposure)
• overall survival during the whole exposure period (day 0–day 33)
• body weights (wet weights) at the end of exposure
• body lengths (total length) at the end of exposure
Differences in survival for each developmental phase analyzed were counted from the observation at the start of the interval to the observation at the end of the interval.
Calculations to determine the NOEC and LOEC were carried out using SAS System
computer software. In all evaluations the statistical unit was the replicate aquarium. All tests were performed one-sided.
The statistical evaluation of fish body weight and length was carried out using Student’s t-test for a simultaneous comparison of the treatment group with the control. For the hatching success, larval survival, juvenile survival and overall survival the Wilcoxon test was used.
The results of the statistical analyses were used to aid in the determination of the NOEC and LOEC. However, scientific judgment was used to determine if statistical differences were biologically meaningful. In this limit test with one test concentration, the NOEC was defined as greater than or equal to the saturation concentration under test conditions when no significant treatment-related adverse effects are observed. The LOEC was defined as greater than the NOEC when no significant treatment-related effects are observed.

Validity criteria fulfilled:

yes

Description of key information

Based on long-term (chronic) toxicity study data, the product is very likely not harmful to fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

> 10 mg/L

Additional information

In a key study (BASF SE, 50F0834/09E041, 2016), the fish early life-stage toxicity test of the test substance using fathead minnow (Pimephales promelas) was conducted for a period of 33 days, following the OECD 210 (2013) and the US EPA Guidelines (OPP Subdivision E 72-4 (a) and OPPTS 850.1400. The overall no observed effect concentration (NOEC) was >10 mg/L (nominal concentration). The overall lowest observed effect concentration (LOEC) was >10 mg/L (nominal concentration). 

 

Furthermore, two valid studies conducted with melamine (CAS 108-78-1) itself are available (Adema 1982, test species: Jordanella floridae, Goodrich, 1984, test species: Oncorhynchus mykiss) that did not reveal regulatorily relevant effects.

In addition to the above mentioned studies, several publications are available that used mixtures of melamine and cyanuric acid to expose different fish species via the diet. Similar to mammals crystal formation in the kidneys could be observed. However, the exposure route via the diet is not regarded as relevant as the compounds are not adsorptive. The effects are not regarded relevant for the hazard assessment.