2(1H)-Naphthalenone, 3,4-dihydro-5-methoxy-

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:

10 July 2006 to 21 July 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Swiss GLP Monitoring Authorities. (issued November 2005)

Specific details on test material used for the study:

- Solubility in water: 1100 mg/L, pH 3.88
- Storage: In the refrigerator at about 4 °C, under nitrogen

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Just before the start of the test duplicate samples were taken from each test solution in medium (without algae), and duplicate samples from the control (without algae). After 72 hours, duplicate samples were taken from each test solution (without algae), and duplicate samples from the control (without algae). For the 72-hour sampling, additional flasks with adequate volumes of the freshly prepared test solutions of all test concentrations and the control were incubated under the same conditions as in the actual test, but without algae.
- Concentrations: Nominal concentrations of 3.2 to 100 mg/L from both sampling times (0 and 72 hours). The samples from the test solutions at nominal concentrations of 0.32 and 1.0 mg/L were not analyzed, since these concentrations were below the determined 72-hour NOEC. From the control samples, only one of the duplicate samples was analyzed at each sampling times.
- Sample storage conditions before analysis: All samples were deep-frozen (at about -20 °C) and protected from light immediately after sampling. Based on pre-experiments (without GLP) for investigation of the storage stability, the test item is sufficiently stable in the test water under these storage conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest nominal test item concentration of 100 mg/L was prepared by dissolving 53.3 mg of the test item completely in 533 mL of test water using ultrasonic treatment (15 minutes) and intense stirring (3 hours at room temperature in the dark). Adequate volumes of this intensively mixed test medium were diluted with test water to prepare the test media with the lower test item concentrations. The test media were prepared just before the start of the test (= addition of algae).
- Evidence of undissolved material:

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG
- Source: Supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany). The algae are cultivated in RCC’s laboratories under standardized conditions according to the test guidelines.
- Age of inoculum: Cells at the start of the test were taken from an exponentially growing pre-culture that was set up 3 days prior to the test under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in synthetic test water, prepared according to the test guidelines.

ACCLIMATION
- Acclimation period: no
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L as CaCO3

Test temperature:

23 °C

pH:

- Start: 8.1 - 8.4
- End: 7.9 - 8.9

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.
- Measured concentrations: n.a., n.a., 0.20, 0.59, 1.1, and 35 mg/L (mean measured test concentrations calculated as the geometric mean over all measurements per test concentration and corrected for % mean recovery of fortified QC samples).

- Recovery: The measured test item concentrations at the start of the test ranged from 24 to 77% of the nominal values (after correction for % mean recovery of fortified QC samples). At test end (t=72 h) the test item was only detected in the highest test concentration solution. Therefore the mean measured test concentrations were calculated as the geometric mean over all measurements per test concentration. These geomean concnetrations varied in the range of 3.3 to 34% of the nominal values. Therefore, the reported biological results are based on the mean measured test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks, covered with glass dishes
- Fill volume: Volumes of 15 mL algal suspension for each replicate continuously stirred by magnetic stirrers.
- Aeration: not reported (assumed 'no')
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 73E+04 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6
- No. of vessels per 'paticle control': 1 (Since particles in the test media might have an influence on the algal counting by the electronic particle counter, additionally one replicate per test concentration was incubated with the test media without addition of algae as ″particle controls″. These additional replicates were incubated and handled in the same manner as the replicates with algae and were used for possible corrections at the algal cell density determinations).

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile purified water
- Culture medium different from test medium: no
- Intervals of water quality measurement: The pH was measured and recorded in each test concentration and the control at the start and at the end of the test. The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The appearance of the test media was also recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: not reported (assumed 'no')
- Photoperiod: continuous illumination
- Light intensity and quality: ca. 8500 Lux (range: 7150 to 9280 Lux). Illumination was achieved by fluorescent tubes (Philips TLD 36W/840), installed about 35 cm above the test flasks

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Cell concentrations (cell densities) were determined after 24, 48 and 72 hours of exposure, by counting with an electronic particle counter (Coulter Counter®, Model ZM), with at least two measurements per sample. For each test flask, the Area under the growth curve (AUC) and Growth rate (r) were calculated according to guideline.
- Other: In addition, after 72 hours exposure, a sample was taken from the control and from a test concentration with reduced algal growth (nominal 10 mg/L). The shape and size of the algal cells were examined microscopically in these samples. This test concentration (nominal 10 mg/L) was chosen since, at the two highest nominal concentrations of 32 and 100 mg/L, the algal cell density was too low for a reliable microscopic examination.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 between the test concentrations was chosen because, according to the results of the range-finding test, the concentration-effect relationship was rather flat and thus, a large concentration range had to be tested
- Range finding study: yes
- Range finding test concentrations: not reported
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate (2005)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no 95% CI determined.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: no 95% CI determined.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes (factor 73 increase in cell density)
- Microscopic examinations: The microscopic examination of the algal cells after 72 hours exposure showed no difference between the algae growing in test medium with reduced algal growth (nominal 10 mg/L) and the algal cells in the control. There were no obvious effects on the shape and size of the algal cells growing in test media containing the test item at up to and including this nominal concentration.
- Any observations that might cause a difference between measured and nominal values: The highest test concentration of 100 mg/L was slightly and homogeneously turbid at test start. At test end, in this test solution a small amount of particles was found lying on the bottom of the test vessel.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h ErC50: 0.78 mg/L
- Other: the toxic performance was within the historical range of the test laboratory (from 1996 to 2005: 72-h EC50: 0.44–1.16 mg/L).

Reported statistics and error estimates:

- EC values: The ErC50 value, the corresponding EC10 value and their 95%-confidence limits were calculated by Probit Analysis. The calculations of the EC values are based on the mean measured test item concentrations. The biological results of the test concentrations of nominal 1.0 and 0.32 mg/L were not taken into account at the Probit Analysis because these test concentrations were below the 72-hour NOEC, and thus not analyzed.
- NOEC: The calculated mean growth rate at the test concentrations were tested for significant differences when compared to the control values by a Dunnett-test.

Table 1: Algal cell density during the 72-h test period

Nominal test conc. (mg/L)	Geomean measured test conc. (mg/L)	Mean density of algal cells (cell number x 10000/mL)
		24 h	48 h	72 h
0 (control)	--	5.38	16.28	72.65
0.32	n.a.	5.52	15.8	73.72
1.0	n.a.	5.63	18.17	87.00
3.2	0.20	5.45	18.48	83.57
10	0.59	4.28	9.63	27.00
32	1.1	3.43	2.37 *	2.35 *
100	35	1.58 *	2.18 *	1.50 *

n.a. = not analysed (below the NOEC)

* Algal counts are corrected for the mean particle densities in the media without algae

 

Table 2: Particle densities in the particle controls (test media without algae)

Nominal test conc. (mg/L)	Geomean measured test conc. (mg/L)	Particle density (particle number x 10000/mL)
		24 h	48 h	72 h
0 (control)	--	0.40	0.50	0.55
0.32	n.a.	0.35	0.25	0.30
1.0	n.a.	0.40	0.25	0.20
3.2	0.20	0.50	0.25	0.40
10	0.59	0.40	0.35	0.30
32	1.1	0.75	3.25	5.45
100	35	4.70	10.70	17.75

 

 

Table 3: Areas under the growth curves (AUC) and percentage inhibition of AUC (IAUC) during the test period

Nominal test conc. (mg/L)	Geomean measured test conc. (mg/L)	24 h		48 h		72 h
		AUC	Inhibition AUC (%)	AUC	Inhibition AUC (%)	AUC	Inhibition AUC (%)
0 (control)	--	53	0	288	0	1332	0
0.32	n.a.	54	-3.2	286	0.8	1336	-0.3
1.0	n.a.	56	-5.9	317	-10.0	1555	-16.8
3.2	0.20	53	-1.7	317	-9.8	1517	-13.9
10	0.59	39	25.0	182	36.8	598	55.1
32	1.1	29	44.4	75	74.1	107	91.9
100	35	7	86.7	28	90.2	48	96.4

 

Table 4: Growth rates r and percentage inhibition of r (Ir) during the test period

Nominal test conc. (mg/L)	Geomean measured test conc. (mg/L)	24 h		48 h		72 h
		r	Ir (%)	r	Ir (%)	r	Ir (%)
0 (control)	--	1.68	0	1.39	0	1.43	0
0.32	n.a.	1.70	-1.7	1.38	1.1	1.43	-0.4
1.0	n.a.	1.73	-3.0	1.45	-3.8	1.49	-4.2
3.2	0.20	1.69	-1.1	1.46	-4.6	1.48	-3.3
10	0.59	1.45	13.4	1.13	19.0	1.10	23.1
32	1.1	1.23	26.6	0.43	69.2	0.28	80.2
100	35	0.45	73.4	0.39	72.2	0.13	90.7

Note: r as 1/day

Validity criteria fulfilled:

yes

Conclusions:

The 72-h ErC50 and 72-h ErC10 values are 0.80 mg/L and 0.033 mg/L, respectively, in freshwater green algae (D. subspicatus). The 72-h NOEC is 0.20 mg/L. All values are based on geomean measured concentrations.

Executive summary:

The toxicity to freshwater green algae (D. subspicatus) was examined in a study according to OECD TG 201 and in compliance with GLP criteria.

Fresh cultures of algae (starting cell concentration of 1E+04 cells/mL) were exposed to nominal test concentrations of 0 (control), 0.32, 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours. Confirmation of test item concentrations in test solutions was performed for the nominal concentrations of 3.2 to 100 mg/L at the start (t=0h) and at the end of exposure (t=72h). The samples from the test solutions of nominal concentrations 0.32 and 1.0 mg/L were not analyzed, since these concentrations were below the determined 72 -hour NOEC. Mean measured concentrations, calculated as the geometric mean over all measurements per test concentration, were determined to be 0.20, 0.59, 1.1, and 35 mg/L. As none of the measured test concentrations was within 80 -120% of nominal at test start and test end, results are expressed as geometric mean measured concentrations (using a value of 0.05 mg/L (i.e. half of the LOQ) for the solutions where no test item was detected at t=72h).

Algal cell concentrations were recorded after 24, 48 and 72 hours exposure and the Area under the growth curve (AUC) and Growth rate (r) calculated. In addition, algal cells were examined microscopically for any abnormalities. Since particles in the test media might have had an influence on the algal counting by the electronic particle counter, additionally, per test concentration, 'particle controls' were included in the test (test solutions without algae). These additional replicates were incubated and handled in the same manner as the replicates with algae and were used for possible corrections at the algal cell density determinations.

At nominal test concentrations of 10, 32, and 100 mg/L, growth rates were significantly inhibited with percentages of 23.1, 80.2, and 90.7, respectively. Based on these findings, the 72-h ErC50 and 72-h ErC10 values were determined to be 0.80 mg/L and 0.033 mg/L, respectively. The 72 -h NOEC was determined at 0.20 mg/L. All values are based on geomean measured concentrations.

The test is valid.

Description of key information

The toxicity to freshwater green algae (D. subspicatus) was examined in a study according to OECD TG 201 and in compliance with GLP criteria.

Fresh cultures of algae (starting cell concentration of 1E+04 cells/mL) were exposed to nominal test concentrations of 0 (control), 0.32, 1.0, 3.2, 10, 32 and 100 mg/L for 72 hours. Confirmation of test item concentrations in test solutions was performed for the nominal concentrations of 3.2 to 100 mg/L at the start (t=0h) and at the end of exposure (t=72h). The samples from the test solutions of nominal concentrations 0.32 and 1.0 mg/L were not analyzed, since these concentrations were below the determined 72 -hour NOEC. Mean measured concentrations, calculated as the geometric mean over all measurements per test concentration, were determined to be 0.20, 0.59, 1.1, and 35 mg/L. As none of the measured test concentrations was within 80 -120% of nominal at test start and test end, results are expressed as geometric mean measured concentrations (using a value of 0.05 mg/L (i.e. half of the LOQ) for the solutions where no test item was detected at t=72h).

Algal cell concentrations were recorded after 24, 48 and 72 hours exposure and the Area under the growth curve (AUC) and Growth rate (r) calculated. In addition, algal cells were examined microscopically for any abnormalities. Since particles in the test media might have had an influence on the algal counting by the electronic particle counter, additionally, per test concentration, 'particle controls' were included in the test (test solutions without algae). These additional replicates were incubated and handled in the same manner as the replicates with algae and were used for possible corrections at the algal cell density determinations.

At nominal test concentrations of 10, 32, and 100 mg/L, growth rates were significantly inhibited with percentages of 23.1, 80.2, and 90.7, respectively. Based on these findings, the 72-h ErC50 and 72-h ErC10 values were determined to be 0.80 mg/L and 0.033 mg/L, respectively. The 72 -h NOEC was determined at 0.20 mg/L. All values are based on geomean measured concentrations.

The test is valid.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.8 mg/L

EC10 or NOEC for freshwater algae:

0.033 mg/L