2-(5-halo-2-hydroxy-aryl)-3H-heteropolycycle-4-one

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:

2019-09-19 to 2020-03

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)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2018-10-25

Analytical monitoring:

yes

Details on sampling:

- Concentrations and sampling method: Samples (2x ~5 mL) were taken prior to start (addition of algae) from freshly prepared test solutions and controls (2 x 6 samples). After 24 and 48 hours, samples were taken from an additional analytical replicate (with algae) from the three highest test concentrations of 25.0 (1:4), 50.0 (1:2) and 100 mg test item/L (saturated solution; 2 x 6 samples). At the end of the test (72 hours), samples were taken from representative replicates per treatment level (2 x 6 samples) and control as well as from the vessels without algae (2 x 5 samples).
- Storage: ≤ -18 °C until analysis

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item (< 63.6 µg/L/20°C), the test item solution was prepared in accordance with the recommendations of OECD 23: The highest test concentration was prepared by dissolving 200.19 mg of the test item in 2 L of sterile growth medium, stirring for 72 h at room temperature (~20°C) and subsequent filtration by a 0.22 µm PES filter. The other test concentrations were obtained by dilution of the highest test concentration (factor 2).
- Controls: Negative control (growth medium)

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Catalog No 61.81 SAG
- Source: SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University of Göttingen, Albrecht von Haller Institut, Untere Karspüle 2, 37073 Göttingen.
- Method of cultivation: The stock cultures are maintained fulfilling the criteria of the OECD guideline. 3 days prior to testing, a pre-culture was established in standard OECD growth medium to obtain exponentially growing algae for the test.

ACCLIMATION
- Pre-culture: Pre-cultures were cultivated for 3 days in the same media as used in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

not reported

Test temperature:

21.75 ± 0.5 °C

pH:

Controls: 7.64 (test start) - 8.17 (mean value of n = 8 controls at test end)
Treatments: 7.58 - 7.64 (test start) to 8.15 - 8.39 (test end)

Dissolved oxygen:

not reported

Salinity:

not reported

Conductivity:

not reported

Nominal and measured concentrations:

Nominal: 0 (control), 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L
Measured at test initiation: Control, 0.693, 1.34, 2.97, 5.14 and 11.5 µg test item/L
Measured at test termination: Control, - (Geometric mean measured concentrations: Control, 0.42, 1.17, 2.39, 4.20, 9.84 µg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL conical glass flasks covered with air-permeable silicone-sponge caps.
- Shaking: At 150 rpm
- Initial cells density: 10,000 cells/mL
- Control end cells density: 964,000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes, OECD medium according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified water processed using an ELGA „PURELAB Ultra“

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Light intensity and quality: 60-120 µE m-2 s-1 (4440 - 8880 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Inhibition of growth, expressed as the logarithmic increase in biomass (average specific growth rate) during the exposure period.
- Determination of cell concentrations: Cell density was determined daily using an electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 1.00, 10.0 and 100 mg test item/L
- Results used to determine the conditions for the definitive study: After filtration, concentrations of 6.70 µg test item/L were measured in the saturated test solution and 5.82 µg test item/L after 72 h of testing (recovery 87 %). In the 1:10 diluted test solution at 0h, a concentration of 0.55 µg/L test item was measured. However, after 72 h of testing, the test item concentration felt below the LOQ (0.5 µg/L).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 9.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 9.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 9.48 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes, factor 104
- Observation of abnormalities (for algal test): Normal and healthy appearance
- Test concentrations and solubility of substance in test medium: The measured concentrations were not within a range of 80 – 120% of the nominal concentrations at test initiation and did not remain stable within ± 20% throughout the test. Therefore, the evaluation was based on the geometric mean measured concentrations of the test item.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 2.78 mg/L (nominal; 95% confidence limits: 2.69 – 2.87 mg/L)
- Other: Tests with the reference substance were routinely performed twice a year. The latest (September 2019) EC50 value of 2.78 mg/L for inhibition of growth rate is in good agreement with the results of an international ring test with an ErC50 of 3.38 ± 1.30 mg/L.

Reported statistics and error estimates:

Calculation of the growth rates, percent inhibition compared to controls of growth rate or sectional growth rate, and yield for the exposure period (72 h) were performed according to the OECD guideline 201. The NOEC values were determined using appropriate statistical methods (Dunnett`s Multiple t-test Procedure, significance level 0.05, one-sided smaller). Since test results showed no inhibition above 50 %, no statistical analysis was performed to determine EC50 and EC10 values together with 95 % confidence intervals. The computer program ToxRat® was used for statistical evaluations.

Table 1: Results of the range finding test

Nominal concentration [mg test item/L]	Measured conc. [µg test item/L]		Effects on algal growth [% inhibition compared to control]
	0 h	72 h	Growth rate	Yield
1.00	<LOQ	<LOQ	-25.1	-128.8
10.0	0.55	<LOQ	-20.6	-97.3
100	6.70	5.82	2.1	9.6

Table 2: Results of the chemical analysis of the test item at test initiation (0 h) and test termination (72 h)

	Test initiation		Test termination
Nominal conc. [µg test item/L]	Measured conc. [µg a.s./L]	% of nominal [%]	Measured conc. [µg a.s./L]	% of nominal [%]	% of initial [%]	Geometric measured conc. [µg test item/L]
Control	< LOQ	n.d.	< LOQ	n.d.	n.d.	n.d.
6250	0.693	0.011	< LOQ^a	0.004	36.1	0.42
12500	1.34	0.011	1.02	0.008	75.6	1.17
25000	2.97	0.012	1.92	0.008	64.6	2.39
50000	5.14	0.010	3.43	0.007	66.8	4.20
100000	11.5	0.012	8.40	0.008	72.9	9.84

^a:            Since the measured test item concentration was below the LOQ, ½ of the LOQ was used for the calculation of the geometric mean measured concentration.

n.d.:       not determined due to mathematical reasons.

LOQ:     0.5 µg/L.

Table 3: Growth rate (G) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Geometric mean measured conc. [µg test item/L]	0-24 h		0-48 h		0-72 h
	G	%I	G	%I	G	%I
Control	1.423	0.0	1.443	0.0	1.546	0.0
0.416	1.318	7.4	1.405	2.6	1.559	-0.9
1.17	1.449	-1.8	1.488	-3.2	1.525	1.3
2.39	1.410	1.0	1.395	3.3	1.540	0.4
4.20	1.424	-0.1	1.567	-8.6	1.565	-1.2
9.84	1.281	10.0	1.438	0.3	1.544	0.1

- negative value indicates increase of the observed parameter compared to the control

Table 4: Yield (Y) and its inhibition relative to control (%I) as computed from the raw data for test intervals selected.

Geometric mean measured conc. [µg test item/L]	0-24 h		0-48 h		0-72 h
	Y	%I	Y	%I	Y	%I
Control	3.2	0.0	17.3	0.0	103.2	0.0
0.416	2.7	13.1	15.6	9.4	106.9	-3.6
1.17	3.3	-5.0	18.8	-8.9	96.8	6.2
2.39	3.2	0.4	15.6	9.7	101.3	1.8
4.20	3.2	-0.9	22.9	-32.4	110.0	-6.6
9.84	2.6	16.7	17.0	1.4	102.2	0.9

Validity criteria fulfilled:

yes

Conclusions:

The impact of the test item on the growth of the freshwater green algae species Pseudokirchneriella subcapitata was determined as follows:

NOEC (growth rate) = >= 9.48 µg/L (geometric mean measured conc. dissolved fraction)
72h-ErC50 (growth rate) > 9.48 µg/L (geometric mean measured conc. dissolved fraction)

All effect concentrations are based on gemetric mean measured concentrations of the dissolved test item concentration.

Executive summary:

The 72-hour toxicity of the test item to the unicellular green alga Pseudokirchneriella subcapitata was determined in a static freshwater system according to OECD 201 (2011). Due to the low water solubility of the test item (< 63.6 µg/L (20°C)), a stock solution was prepared using 100 mg test item/L test medium and stirred over 72 hours to achieve the maximum water solubility (in accordance with the OECD series on testing and assessment no. 23). Based on a serial dilution by factor 2, nominal concentrations of 0 (control), 6.25 (1:16), 12.5 (1:8), 25.0 (1:4), 50.0 (1:2) and 100 mg test item/L (saturated solution) were tested. The dissolved test item concentrations in all test solutions were measured by UHPLC-HRMS (LOQ = 0.5 µg test item/L) before start (0 h) and at test end (72 h). Due to a decrease in test item concentrations over the course of the test, the evaluation of results was based on geometric mean measured values (0 (control), 0.416, 1.17, 2.39, 4.20 and 9.84 µg test item/L).

Concentration-dependent inhibiting effects by the test item on the growth of the freshwater green algae over the range of the tested concentrations were not observed. The 72h ErC50 and EyC50 were determined to be > 9.84 µg test item/L (corresponding to the maximum solubility of the test item in test solution, equivalent to a nominal loading of 100 mg/L). The NOEC (growth rate and yield) was determined to be ≥ 9.84 µg test item/L. All validity criteria of the test were met.

Description of key information

The impact of the test item on the growth of the freshwater green algae species Pseudokirchneriella subcapitata was determined as follows:

NOEC (growth rate) = >= 9.48 µg/L

72h-ErC50 (growth rate) > 9.48 µg/L

All effect concentrations are based on gemetric mean measured concentrations of the dissolved test item concentration.