Hexahydro-1,3,5-trimethyl-1,3,5-triazine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2007 to 17 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted in accordance with international guidelines in a GLP testing laboratory.

Qualifier:

according to guideline

Guideline:

other: ISO TC147/SC5/WG2 protocol by Thompson (1990): Water Quality: Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)

Deviations:

no

GLP compliance:

yes

Remarks:

GLP compliance statement only

Analytical monitoring:

no

Details on sampling:

N/A

Vehicle:

no

Details on test solutions:

The dilution water used for culturing the copepods used in this study and for the test was supplied by Guernsey Sea Farms and was delivered on 13 March 2007. The dilution water was stored in the dark at approximately 15oC within the testing facility. Immediately before use, it was aerated until the dissolved oxygen concentration reached saturation (>80 % ASV) and the pH stabilised at 8.0 ± 0.3.The behaviour of the test item was examined in seawater according to the method described in the Harmonised Offshore Chemical Notification Format (OSPAR 1995). A nominal 424 mg/L solution of hexahydro-1,3,5-trimethyl-1,3,5-triazine (1000 mg/L) was prepared in dilution water, shaken vigorously and allowed to stand for 1 hour.A clear colourless liquid was obtained. Test concentrations were prepared by addition of specified quantities of the sample to dilution water to obtain the required concentrations.A preliminary study had identified the 48 hour LC50 as being between 4.24 and 42.4 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine and therefore definitive test concentrations were prepared as 0 (Control), 1.908, 4.24, 9.328, 19.08, 42.4 and 93.28 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine.

Test organisms (species):

other: Acartia tonsa

Details on test organisms:

TEST SPECIESTest organism: Acartia tonsaSource: Guernsey Sea FarmsDate organisms delivered to laboratory: 13 March 2007Age of copepods at start of test: 16 ± 1 dayLife stage: AdultHolding conditions of culture prior to despatch:Temperature: 20 ± 1 oCSalinity: 33 ± 2 pptFeeding regime: Mixed algal diet as recommended by Guernsey Sea Farms.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

N/A

Test temperature:

Refer to Table 1.

pH:

Refer to Table 1.

Dissolved oxygen:

Refer to Table 1.

Salinity:

Refer to Table 1.

Nominal and measured concentrations:

Nominal concentrations: 1.908, 4.24, 9.328, 19.08, 42.4 and 93.28 mg/L with an untreated control.

Details on test conditions:

Four 25 ml aliquots of each test concentration were measured out into 4 replicate 50 ml crystallising dishes for each concentration. A glass tube with a 5 mm bore was used to transfer 5 copepods into each test container. Four control vessels were prepared as 25 ml of dilution water each containing 5 copepods. The reference toxicant, 3,5 Dichlorophenol (DCP), was used to check that the copepod sensitivity was within acceptable limits. Four replicate test vessels of a 1 mg/L concentration of 3,5 DCP were prepared with 5 copepods in each. A mortality of between 20 to 80 % is considered acceptable for Acartia exposed to 1 mg/L of 3,5 DCP for 48 hours. The test vessels were placed into an incubator at a temperature of 20 ± 2oC with a light cycle of 16 hours light and 8 hours dark.The pH value (to 0.1), dissolved oxygen (to 1% ASV), temperature (to 0.5oC) and salinity were measured on the dilution water immediately prior to initiating the test, and were measured at the end of the test for both the control and the lowest concentration causing 100 % mortality of exposed copepods. A refractometer was used to measure the salinity. The numbers of dead copepods were recorded after 24 and 48 hour exposure periods. Where possible, the LC50 values were estimated and 95 % confidence limits calculated using ToxCalc version 5 "Comprehensive Toxicity Data Analysis and Database Software".

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

26.288 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL = 23.744 to 29.256 mg/L

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

20.352 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95 % CL = 16.96 to 23.744 mg/L

Details on results:

None of the 20 control copepods died during the study.

Results with reference substance (positive control):

The 1mg/L 3,5-DCP reference substance exhibited 70 % mortality which is within the required range (20-80 %) required for a valid test.

Reported statistics and error estimates:

LC50 values determined by the Spearman-Karber method.NOEC and LOEC values determined by Fishers Exact Test.

Table 1. Water Quality Parameters

Water Quality Parameter	Before Test		End of Test
	Control	42.4 mg/L	Control	42.4 mg/L
pH	8.2	8.5	7.9	8.3
DO (% ASV)	100	100	98	98
Temperature (^oC)	20.0	20.0	20.0	20.0
Salinity (‰)	32	32	33	33

Table 2. Cumulative Percent Mortalities

Nominal Concentration (mg/L)	Number Dead		% Mortality
	24 hours	48 hours	24 hours	48 hours
Control	0	0	0	0
1.908	0	0	0	0
4.24	0	0	0	0
9.328	0	0	0	0
19.08	2	9	10	45
42.4	20	20	100	100
93.28	20	20	100	100
3,5 Dichlorophenol (1mg/L)	4	14	20	70

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour LC50 value of Amietol M33 to Acartia tonsa was estimated by ToxCalc analysis to be 20.352 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine as determined by the Spearman-Karber method. The no-observed effect concentration (NOEC) after 48 hours was 9.328 mg/L.

Executive summary:

Substance: 42.4 % hexahydro-1,3,5 -trimethyl-1,3,5 -triazine

Chemex reference: Sample: ECO020727; Study: ENV8006

Test species: Acartia tonsa

Test type: Acute toxicity: 48 hour LC50

Test Conditions: Static test conditions according to SOP E207 based upon ISO TC147/SC5/WG2 protocol by Thompson (1990): Water Quality: Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea).

Test Period: 15 March 2007 to 17 March 2007

Test performed at: Chemex Environmental International Limited, Unit J, Broad Lane Industrial Estate, Cottenham, Cambridge, CB24 8SW, UK

Results:

Period of Exposure (hours)	LC50Value (mg/L)	95 % Confidence Limits (mg/L)
24	26.288	23.744 - 29.256
48	20.352	16.96 - 23.744

Description of key information

48 h LC50 (Acartia tonsa) = 20.352 mg/L, ISO TC147/SC4/WG2, (Fisher, 2007)

Key value for chemical safety assessment

Marine water invertebrates

Marine water invertebrates

Effect concentration:

20.352 mg/L

Additional information

A single key study was submitted for this endpoint, conducted in accordance with ISO Guideline TC147/SC5/WG2. The 48h LC50 to Acartia tonsa was estimated to be 20.352 mg/L (Fisher, 2007).