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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2007 to 22 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:

ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)

Version / remarks:

Draft

Deviations:

no

GLP compliance:

yes

Remarks:

GLP compliance statement only.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):N/A

Analytical monitoring:

no

Details on sampling:

N/A

Vehicle:

no

Details on test solutions:

N/A

Test organisms (species):

Skeletonema costatum

Details on test organisms:

Species: Skeletonema costatum strain: 1077/5Source: Culture Collection of Algae and Protozoa, Dunstaffnage Marine Laboratory, ScotlandCulture conditionsTemperature: 20 ± 1oCIllumination: 6000 - 10000 lux continuous white lightShaking: 200 rpmCulture media: Natural seawater with added nutrients according to the ISO standard, with concentrations of iron and EDTA at the levels recommended by VKI (Danish Water Qualit yInstitute) - See attached study report.

Test type:

static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

N/A

Hardness:

N/A

Test temperature:

Refer to table 1.

pH:

Refer to table 1.

Dissolved oxygen:

Not reported.

Salinity:

Not reported.

Nominal and measured concentrations:

Nominal concentrations: 0.424, 0.933, 1.908, 4.24 and 9.328 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine and an untreated control.

Details on test conditions:

PREPARATION OF TEST SOLUTIONSThe 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 hexahydro-1,3,5-trimethyl-1,3,5-triazine solution was prepared in dilution water, shaken vigorously and allowed to stand for 1 hour.A clear colourless solution was obtained. Test concentrations were prepared by mixing specified quantities of the sample with culture medium to obtain the required concentrations.A preliminary study had identified the 72 hour EC50 as being between 0.424 and 4.24 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine and therefore definitive test concentrations were prepared as 0, 0.424, 0.933, 1.908, 4.24 and 9.328 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine.TEST METHODS AND CONDITIONSStatic test conditions according to ISO draft method "Water Quality Marine algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum.Chemex SOP reference: E209 "Marine Algal Growth Inhibition test with Skeletonema costatum and Phaeodactylum tricornutum."Test Period: 19 March 2007 to 22 March 2007Test Volume: 200 mlTest Vessel: 250 ml conical flaskReplication: 6 control flasks, 3 replicates at each test concentrationSeawater: Collected on 6 December 2006 from the CEFAS laboratories at Burnham-on-Crouch directly from the River Crouch estuaryAlgal Test Inoculum: From a pre-culture growing in exponential phase. Inoculum level adjusted to give an initial cell density of 1 x 10^4 cells/mL.Test Conditions:Temperature: 20 ± 1oCIllumination: 6000 - 10000 lux continuous white lightShaking: 200 rpmWater Quality Measurements: The pH (to 0.1) and temperature (to 0.5oC) were measured on the pooled replicates for each test and control solution immediately prior to initiating the test and at the end of the full 72 hour test period.Observations: The cell density was determined at 24, 48 and 72 hours on a small volume removed from each replicate flask. The cell counts were made using a haemocytometer and microscope.Calculation and Expression of Results: Growth curves for each test concentration were plotted as logarithm of the mean cell density against time. The inhibition of growth was calculated using both the biomass (area under the growth curve) and the growth rate method. Where possible, the EC50 values were estimated graphically and 95 % confidence limites calculated according to the method of ToxCalc Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software", copyright 1994-1996.

Reference substance (positive control):

yes

Remarks:

3, 5-dichlorophenol

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.721 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL = 0.636 to 0.7632 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.145 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL = 1.060 to 1.230 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.187 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL = 1.060 to 1.314 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.908 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL = 1.781 to 2.078 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.933 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The growth curves in the attached study reports suggest that at a concentration of 0.424 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine growth of the algae was good, higher that that of the controls. At 0.933 mg/L hexahydro-1,3,5-trimethyl-1,3,5-triazine growth was slightly inhibited, showing a clear dose response to the test material. However at 1.908 mg/L growth was poor and at concentrations above this, no significant growth occured throughout the test period. The inhibition values in table 2 support these observations.

Results with reference substance (positive control):

The algae were exposed to a 1.5 mg/L concentration of 3,5 DCP for 72 hours under test incubation conditions and therefore the 69 % inhibition by growth rate represents an acceptable level of sensitivity (i.e. 72 hour inhibition was within the range of 20 to 80%).

Reported statistics and error estimates:

EC50 values for biomass integral determined using the Maximum Likelihood-Probit Method.EC50 values for growth rate were determined using the Spearman-Karber Method.

Table 1. Water Quality Data

Nominal Concentration (mg/L)	Mean pH Values		Temperature (^oC)
	Start of Test	End of Test	Start of Test	End of Test
0	7.6	8.3	22.0	21.0
0.424	7.6	8.5	22.0	21.0
0.933	7.6	8.6	22.0	21.0
1.908	7.7	8.2	22.0	21.0
4.24	7.7	8.0	22.0	21.0
9.328	7.7	8.0	22.0	21.0

Table 2. Percent Inhibition by Biomass Integral and Growth Rate

Nominal Concentration (mg/L)	% Inhibition by biomass integral		% Inhibition by Growth Rate
	48 hours	72 hours	48 hours	72 hours
0.424	14	-19	2	-5
0.933	71	32	37	4
1.908	98	90	78	46
4.24	100	100	100	99
9.328	100	100	98	100

Note: Negative inhibition values indicate an increase in growth compared with controls.

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC50 of hexahydro-1,3,5-trimethyl-1,3,5-triazine to Skeletonema costatum was estimated as 1.145 mg/L using the biomass integral and 1.908 mg/L by growth rate calculation. The 48 hour EC50 was 0.7208 mg/L by biomass integral and 1.187 mg/L by growth rate.The NOEC is either 0.424 mg/l or 0.933 mg/l. In both cases, it would be in the range of >0.1 to =<1mg/l. In addition, the substance “is considered to meet the criteria of "readily biodegradable"

Executive summary:

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

Chemex reference: Sample ECO020727; Study ENV8007

Test Species: Skeletonema costatum, strain 1077/5

Test type: Acute toxicity, 72 hour EC50

Protocol: Static test conditions according to SOP E209 based on ISO/DIS BS EN ISO 10253: 1998 "Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum.

Test Period: 19 March 2007 to 22 March 2007

Test Concentrations: 0, 0.424, 0.933, 1.908, 4.24 and 9.328 mg/L hexahydro-1,3,5 -trimethyl-1,3,5 -triazine

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

Result:

Period of Exposure (hours)	EC50 value by biomass integral (mg/L hexahydro-1,3,5 -trimethyl-1,3,5 -triazine )	EC50 value by growth rate (mg/L hexahydro-1,3,5 -trimethyl-1,3,5 -triazine )
48	0.721	1.187
72	1.145	1.908

The NOEC is either 0.424 mg/l or 0.933 mg/l. In both cases, it would be in the range of >0.1 to =<1mg/l. In addition, the substance “is considered to meet the criteria of "readily biodegradable"

The second adaptation to technical and scientific progress (ATP) to the CLP Regulation ((Commission Regulation (EU) No 286/2011 of 10 March 2011) introduced additional criteria for substances to be classified into the aquatic hazardous categories based on the chronic toxicity data. According to the amended criteria rapidly degradable substances for which adequate chronic toxicity data are available will be classified as aquatic Category Chronic 3 when chronic NOEC for algae or other aquatic plants is >0.1 to =<1mg/l (see Tables 4.1.0.(b) and 4.1.4 of Part 4 of Annex I to the CLP Regulation, as amended by Commission Regulation (EU) No 286/2011 of 10 March 2011)

Description of key information

72 h EC50 (growth rate, Skeletonema costatum) = 1.908 mg/L, ISO/DIN BS EN 10253, Fisher (2007)

72 h NOEC (growth rate, Skeletonema costatum) = 0.933 mg/L, ISO/DIN BS EN 10253, Fisher (2007)

Key value for chemical safety assessment

EC50 for marine water algae:

1.908 mg/L

EC10 or NOEC for marine water algae:

0.933 mg/L

Additional information

A single key study was conducted, assessing the growth inhibition of the substance to the marine algae Skeletonema costatum, conducted in accordance with ISO/DIN BS EN 10254. The 72 h EC50 (growth rate) was estimated to be 1.908 mg/L and the 72 h NOEC (growth rate) was determined to be 0.933 mg/L (Fisher, 2007).