1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

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:

16 June 2022 to 30 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test concentrations along with the negative control and the vehicle control were analyzed for test item at the beginning and at the end of the test.
For analysis, duplicate composite samples were drawn from prepared test concentrations, and from the negative and the vehicle controls.
Analysis was conducted using a validated analytical method. The criteria for acceptance of the results of test concentration analysis were 80 to 120% of nominal concentration with ≤20% RSD of analyzed concentration.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

Test item stock of 1 mg/mL was prepared by mixing 1000 mg of test item initially mixed with 0.1 mL of acetone and made up the volume to 1000 mL using growth medium in a volumetric flask and sonicated for 30 minutes. Growth medium with 0.01% acetone was prepared and used for preparation of test concentrations. 0.7 mL of acetone was mixed and made up to 7 L using growth medium in a pre-marked glass container. To prepare test concentrations of 50, 80, 128, 204.8 and 327.7 mg/L, 50, 80, 128, 204.8 and 327.7 mL of test item stock was mixed and made up to 1 L in a volumetric flask using growth medium with 0.01% acetone, respectively.
1 L of growth medium containing 0.01% acetone was used as vehicle control (G2).
For negative control (G1), required volume of algal cell suspension was made up to 1 L using growth medium.
Algal cell suspension of 3.6 mL was added before final volume of 1 L was made up for each test concentration, negative and vehicle control to achieve an initial cell density of 1x10^4 cells/mL.
Each replicate was prepared by dispensing 150 mL of the previously prepared test solutions.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) ATCC® 22662. This algal species was used as the test system as it is a recommended algal species as per the guideline.
- Source: American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108, USA

ACCLIMATION
- Culturing media and conditions: Algal cells were maintained at the test facility through sub-culturing once in 5 to 10 days from the continuous culture into fresh medium in a conical flask. These conical flasks were incubated between
21 and 25°C (±2°C) under a cool white fluorescent continuous illumination of 8000±1600 Lux.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.1 to 22.6 °C

pH:

8.11 to 8.18

Nominal and measured concentrations:

Nominal concentrations: 50 mg/L, 80 mg/L, 128 mg/L, 204.8 mg/L and 327.7 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks, closed using cotton.
- Initial cells density of inoculum at start of exposure: 240 x10^4/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per negative control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: Yes.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterilized water.
- Culture medium: Growth medium (test medium) was algal medium as described in Annex 3 of the OECD test guideline 201 (OECD 2006).

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the medium was 8.19 during definitive test. This medium was equilibrated with carbon dioxide by keeping it in laminar hood for two hours.
- Light intensity and quality: Cool white fluorescent continuous illumination of 8000±1600 Lux.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The cell growth was measured at 24, 48 and 72 hours after the initiation of the test using a haemocytometer (microscopic observation).
- Other: The concentration-effect relationship was determined using two factors namely growth rate and yield at the end of the test (72 hours).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6
- Range finding study: A range finding test was conducted to select the test concentrations for the definitive test with a negative control, vehicle control and test item concentrations at 0.01, 0.1, 1, 10 and 100 mg/L. Each group had a single replicate.
- Results used to determine the conditions for the definitive study: During range finding study, the test item exhibited 0, 0, 0, 0 and 20.02 percent cell reduction at the tested concentrations of 0.01, 0.1, 1, 10 and 100 mg/L at 72 h.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

170.37 mg/L

95% CI:

>= 157.468 - <= 184.327

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

108.119 mg/L

95% CI:

>= 99.931 - <= 116.977

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

85.243 mg/L

95% CI:

>= 78.788 - <= 92.227

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

95.183 mg/L

95% CI:

>= 86.498 - <= 104.739

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

61.649 mg/L

95% CI:

>= 56.024 - <= 67.838

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

49.126 mg/L

95% CI:

>= 44.644 - <= 54.058

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The average cell biomass was 80.75, 51.67, 22.25, 6.17 and 1.42 (x 10^4 cells/mL) at the tested concentrations 50, 80, 128, 204.8 and 327.7 mg/L respectively.
The concentration-effect relationship was determined using two factors namely growth rate and yield at the end of the test (72 hours). The inhibition of growth rate was 0, 10.11, 29.30, 58.54 and 92.01% and the inhibition of yield was 0.0, 36.30, 73.28, 93.50 and 99.48% at the nominal concentrations 50, 80, 128, 204.8 and 327.7 mg/L respectively, when compared to the vehicle control. Observed cells were found to be morphologically normal.
The test item concentration analysis in all test concentrations showed that the mean percent recovery with the nominal concentration was from 94.592 to 107.275% at the start of the test (RSD was 0.085 to 0.491%) and 81.644 to 95.151% at the end of the test (72 hours) (RSD was 0.262 to 0.777%) indicating that the results were within the acceptable limit (80 to 120% of the nominal concentration with an RSD of < 20%).

The validity criteria of the test are fulfilled as:
- There was an increase in cell concentration of the negative control culture by a factor of 79.54, which is more than the required factor limit of at least 16 at the end of the test.
- The mean coefficient of variation for section-by-section specific growth rates in the negative control cultures during the test was 23.94%, which is within the required limit of 35%.
- The coefficient of variation of average growth rate between replicate cultures of negative control was 0.28%, which is within the required limit of 7%.

Results with reference substance (positive control):

The test with reference substance Potassium dichromate (K2Cr2O7) has met all the validity criteria.
ErC50 (growth rate) = 0.6500 mg/L (95% Cl: 0.28890-1.38270)
EyC50 (yield) = 0.2130 mg/L (95% Cl: 0.16640-0.27350)

Reported statistics and error estimates:

The EC10, EC20 and EC50 with 95% confidence limits will be determined by calculating the ErC and EyC by Probit analysis of Finney (1978) using a validated computer program.
For estimation of the NOEC, for effects of the test item on growth rate, analysis of variance (ANOVA) technique was used.

Validity criteria fulfilled:

yes

Conclusions:

The test item AP 729 was tested for its effect on growth of freshwater unicellular green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) for 72 hours. The following EC50 values were found: ErC50 = 170.37 mg/L, EyC50 = 95.183 mg/L.

Executive summary:

The effects of AP 729 were tested according to OECD Test Guideline 201 on the growth of freshwater unicellular green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) for 72 hours.

The algae were exposed to the test item at the test concentrations of 50, 80, 128, 204.8 and 327.7 mg/L (factor of 1.6) along with a negative control and vehicle control. Six replicates were maintained for the negative control and the vehicle control, and three replicates for the test concentrations. The cell growth was measured at 24, 48 and 72 hours after the initiation of the test using a haemocytometer (microscopic observation).
The concentration-effect relationship was determined using two factors, namely growth rate and yield at the end of the test (72 hours). The inhibition of growth rate was 0, 10.11, 29.30, 58.54 and 92.01% and the inhibition of yield 0.0, 36.30, 73.28, 93.50 and 99.48% at the nominal concentrations 50, 80, 128, 204.8 and 327.7 mg/L respectively, when compared to the vehicle control. 
The test item concentration analysis in all test concentrations showed that the mean percent recovery with the nominal concentration was from 94.592 to 107.275% at the start of the test (RSD was 0.085 to 0.491%) and 81.644 to 95.151% at the end of the test (72 hours) (RSD was 0.262 to 0.777%) indicating that the results were within the acceptable limit (80 to 120% of the nominal concentration with an RSD of < 20%).
Observed cells were found morphologically normal in all the tested concentrations.

The endpoint values for growth rate and yield at 72 hours, based on nominal concentration, are presented below in Table 1:

 

Table 1

At 72 hours	EC values (mg/L)	95% Cl
ErC50	170.37	157.468-184.327
ErC20	108.119	99.9315-116.9766
ErC10	85.243	78.788-92.2266
EyC50	95.183	86.498-104.7392
EyC20	61.6487	56.02383-67.83842
EyC10	49.126	44.64367-54.05836
LOEC	80	NA
NOEC	50	NA

 

Description of key information

The effects of AP 729 were tested according to OECD Test Guideline 201 on the growth of freshwater unicellular green alga Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) for 72 hours.

The algae were exposed to the test item at the test concentrations of 50, 80, 128, 204.8 and 327.7 mg/L along with a negative control and vehicle control. Six replicates were maintained for the negative control and the vehicle control, and three replicates for the test concentrations. 

The concentration-effect relationship was determined using two factors, namely growth rate and yield at the end of the test.

The validity criteria of the test were fulfilled. 

The following EC50 values were found: ErC50 = 170.37 mg/L, EyC50 = 95.183 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

170.37 mg/L

EC10 or NOEC for freshwater algae:

50 mg/L