Reaction product of salicylaldehyde and formaldehyde and phenol

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:

15 May 2012 to 18 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Algal Growth Inhibition Test stipulated in the "Testing Methods for New Chemical Substances" of Japan

Version / remarks:

March 31, 2011; No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No.5, Manufactuiing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Envirornnent, Japan

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 23 (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures)

Version / remarks:

2000

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All
- Sampling method: At the start of exposure the solution was sampled from the container for preparation and at the end of exposure the mixed solution was taken out with equal volume of the test solution from the test vessels in each test level. 50 - 60 mL was taken.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The ground test sample was dissolved in acetone to prepare acetone solution of 1000 mg/L. This solution was diluted with acetone as appropriate to prepare the test material solution, whose concentration was 100 times of each test concentration as test material solution. The test material solution was added into the preparation container, and inner of the container was coated with the test material by evaporating the acetone under a stream of nitrogen. To remove acetone completely, the container was heated on a hot plate (approximately 40 °C) for approximately 10 minutes. Medium was added into each container to prepare each test concentration (nominal concentration) and stirred for 24 hours. The prepared solution was used as the test solution and divided into each test vessel.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ATCC 22662
- Source: American Type Culture Collection
- Method of cultivation: Passage cultured under sterile conditions in the testing laboratory

CULTURE CONDITIONS
- Culturing media and conditions same as test or not: yes
- The algae were counted in pre-culture incubated under the same conditions as the test for 3 days as inoculums, and were added to test vessels to bring the initial cell concentration to 0.75 x 10^4 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.8 - 23.2 °C

pH:

7.8-8.0

Nominal and measured concentrations:

Nominal: 0.058, 0.16, 0.46, 1.3, 3.6 and 10 mg/L
Geometric mean measured: 0.061, 0.15, 0.46, 1.3, 3.6 and 9.9 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilised 500 mL Erlenmeyer flask (with gas-permeable Silicosen)
- Material, size, headspace, fill volume: 100 mL/test vessel
- Flasks were incubated with rotary shaking (approximately 100 rpm)
- Initial cells density: The algae were counted in pre-culture incubated under the same conditions as the test for 3 days as inoculums, and were added to test vessels to bring the initial cell concentration of 0.75 x 10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium (OECD TG 201; March 23, 2006) prepared with purified water was used. Components: H3BO3 0.185 mg/L, CuCl2.2H2O 0.00001 mg/L, MnCl2.4H2O 0.415 mg/L, CaCl2.2H2O 18.0 mg/L, ZnCl2 0.00300 mg/L, NH4Cl 15.0 mg/L, FeCl3·6H2O 0.0640 mg/L, KH2PO4 1.60 mg/L, Na2EDTA.2H2O 0.100 mg/L, NaHCO3 50.0 mg/L, CoCl2.6H2O 0.00150 mg/L, MgCl2.6H2O 12.0 mg/L, Na2MoO4.2H2O 0.00700 mg/L and MgSO4.7H2O 15.0 mg/L.
- Intervals of water quality measurement: pH was measured in another solution sampled from the container for preparation (at the start of exposure) and in one test vessel in each test level at the end of exposure. Culture temperature was measured once a day during the exposure. Light intensity was also measured once a day during the exposure in the incubator.

OTHER TEST CONDITIONS
- Sterile test conditions: yes/no
- Photoperiod: continuous
- Light intensity and quality: Nominal 90 μE·m^-2·s^-1 ± 20% (within ± 15% from the average light intensity). Continuous illumination provided by fluorescent lights with wavelength range of 400-700 nm.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Biomass (cell concentration) was measured every 24 hours after the start of the exposure (the blank correction was conducted by measuring the value of the blank solution). Cells were counted with a particle counter; Model COULTER Zl (Beckman Coulter, Inc.)
- An observation of cell condition was performed in one vessel in each test level at the end of exposure.
- Other: The appearance of test solutions was observed at the start and end of exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: a geometric series with a factor of 2.8
- Range finding study: The test concentrations and the factor were decided based on the results of preliminary studies.

TREATMENT OF THE RESULTS
- The results of the study were estimated by nominal concentration, because the measured concentration in the test solution during the exposure was within the range of ± 20% of the nominal concentration.
- Calculation of concentration-inhibition rates: The mean value of biomass for each test level was plotted against time to produce growth curves. Using this curve, inhibition rates were calculated comparing with control values on growth rate.
- Comparison of growth rates: The specific growth rate for a specific period was calculated as the logarithmic increase in biomass according to the following formula:
µ(i-j) = [ (lnNj – lnNi) / (tj – ti) ]
where
μ(i-j) = Specific growth rate from time i to j (normally d^-1)
Ni = Value of cell concentration (cells/mL) at ti: Set value of cell concentration was used at the start of the exposure (t0).
Nj = Value of cell concentration (cells/mL) at tj
ti = Time (d) of ith measurement after beginning of exposure
tj = Time (d) of jth measurement after beginning of exposure
- Specific growth rate over the exposure duration (0-72 h) was calculated for determination of EC50. In control, specific growth rates for section-by-section were calculated for check of validity of the test.
- The percentage inhibition for each exposure level was mean value of the percent inhibition in average specific growth rate for a replicate (Iμ) in test level. The percent inhibition (Iμ) was calculated from mean value for average specific growth rate in the control (μC), average specific growth rate for each replicate in exposure level (μT), and the following formla:
Iµ = [(µC - µT) / µC] x 100

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence interval: 3.5-4.3 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

OBSERVATION AND MEASUREMENT OF TEST SOLUTION
- Appearance of test solution: At the start of exposure, test solutions of all exposure levels were colourless and clear. At the end of the exposure, the test solution in 10 mg/L exposure level did not change, and due to the algae growth, that in 3 .6 mg/L exposure level was light green, and those in the other exposure levels were green. The solution of control was colourless and clear at the start of exposure, and it was green at the end of exposure due to the algal growth.
- Water quality and environmental conditions: The measured values of pH were 7.8-8.0. Culture temperatures in incubator were 22.8-23.2°C and light intensities were 88-95 μE·m^-2·s^-1.
- Concentration of test material in test solution: The measured concentrations of the test material in the test solutions at the start of exposure were 0.059-9.7 mg/L which were 93-101% of the nominal concentration, and those at the end of exposure were 0.063-10 mg/L which were 92-109% of the nominal concentration. Therefore, the test material concentrations in the test solution were maintained within ± 20% of the nominal concentrations.

ErC50
- ErC50 of the test material based on the growth rate was 3.9 mg/L (95% confidence interval: 3.5-4.3 mg/L).

GROWTH CURVES, CELL OBSERVATIONS AND NOEC
- At the 10 mg/L level, algal growth was inhibited remarkably during the exposure. The algal growth at the 3.6 mg/L level was logarithmic, although growth inhibition was found. The algal growth at the 1.3, 0.46 and 0.058 mg/L levels was close to that in the control, although growth inhibition was found. The algal growth at the 0.16 mg/L level was the same as that in the control level.
- The following results of cell observation were based on the comparison with the control: Bloat cells were observed a little more at the 10 mg/L level, and slightly bloat cells were observed a little more at the 3.6 mg/L level. The conditions of cells in the other exposure levels were same as the control. In the control, the condition of cells was not abnormal. In the growth rate, statistically significant differences were observed in 3.6, 1.3, 0.46 and 0.058 mg/L levels. There was no significant difference at the 0.16 mg/L level. There was some degree of concentration-response relativity among the 0.16-3.6 mg/L levels. Inhibition rate of 0.058 mg/L level was 3.4%, which was low, and the inhibition can not be denied owing to the environmental conditions.
- Therefore, with the consideration of the results of cell observation, NOEC based on growth rate was estimated as 0.16 mg/L.

VALIDITY OF THE TEST
- Growth of the control: The cell in the control grew exponentially during the exposure. At the end of exposure, it increased to 103 or more times of the number of initial cells in the control. This meets the validity of the test: the cell growth in control should have increased by a factor of at least 16 times at 72 hours after the start of the exposure.
- The specific growth rates of section-by-section in the controls: The mean coefficient of variation for section-by-section specific growth rate in the controls was 6.6%. It meets the validity of the test: the mean coefficient of variation in the control must not exceed 35%.
- The specific growth rates in replicate controls: The coefficient of variation of specific growth rate in replicate controls was 0.97%. It meets the validity of the test: the coefficient of variation in controls must not exceed 7%.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.75 mg/L
- This value was within the normal range of the reference substrance in this laboratory (mean ± 2 S.D.) [mean ± S.D.: 0.94 ± 0.17 mg/L (n=19)].

Reported statistics and error estimates:

Estimation of EC50
- The percentage inhibition in each exposure level was plotted on semi-logarithmic graph against the corresponding concentration. Linear regression analysis (least square method) was carried out using the data within the range showing linearity visually to estimate the EC50, which was estimated from the intercept of the regression line with the parallel drawn to the abscissa at 50% of inhibition. The EC50 was denoted as ErC50 based on growth rate. The EC50 was conducted using computer program (running on Microsoft Excel) constructed by the laboratory.

Estimation of NOEC
- Regarding the growth rate, Bartlett's test was done to determine the homogeneity of variance for the data. Then one-way analysis of variance and Dunnett's multiple comparison test were used to determine the significant difference between the control level and exposure levels. The statistical analysis was conducted using computer program (running on Microsoft Excel) constructed by the laboratory. NOEC was determined by the results of statistical analysis and cell condition.

Table 1: Measured Concentrations of Test Material in Test Solutions

Nominal Concentration (mg/L)	Measured Concentration (mg/L) (Percentage of Measured Concentration Versus Nominal Concentration, %)
	Start of the Test	End of the Test	Geometric Mean
Control	n.d.	n.d.	-
0.058	0.059 (101)	0.063 (109)	0.061 (105)
0.16	0.15 (93)	0.15 (92)	0.15 (92)
0.46	0.46 (99)	0.47 (103)	0.46 (101)
1.3	1.3 (97)	1.3 (101)	1.3 (99)
3.6	3.6 (100)	3.5 (97)	3.6 (99)
10	9.7 (97)	10 (101)	9.9 (99)

n.d.: <0.0110 mg/L

Table 2: Growth Rate and Inhibition

Nominal Concentration (mg/L)		Growth Rate (0-3 d)	Inhibition Rate (%)
Control	Mean	1.57	-
	S.D.	0.0151	-
0.058	Mean	1.52	3.4
	S.D.	0.00818	0.52
0.16	Mean	1.56	0.58
	S.D.	0.0114	0.73
0.46	Mean	1.48	5.7
	S.D.	0.0281	1.8
1.3	Mean	1.48	5.4
	S.D.	0.00675	0.43
3.6	Mean	0.951	39
	S.D.	0.0288	1.8
10	Mean	0.0782	95
	S.D.	0.00897	0.57

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the ErC50 of the test material based on the growth rate was 3.9 mg/L (95 % confidence interval: 3.5-4.3 mg/L) and the NOEC was 0.16 mg/L.

Executive summary:

The toxicity of the test material to algae was investigated in accordance with the standardised guideline OECD 201 and Japanese guidelines, under GLP conditions.

The objective of the study was to determine the 0-72-hour median effective concentration (EC50) and no observed effect concentration (NOEC) by conducting an algae growth inhibition test of the test material in Pseudokirchneriella subcapitata.

The algae was exposed to the test material at nominal concentrations of 0.058, 0.16, 0.46, 1.3, 3.6 and 10 mg/L. The test material concentrations were analysed at the start and end of the test with high performance liquid chromatography. The measured concentrations of the test material in the test solution were within the range of ± 20% of nominal concentrations.

ErC50 of the test material based on the growth rate was found to be 3.9 mg/L (95% confidence interval: 3.5-4.3 mg/L). At the 10 mg/L level, algal growth was inhibited remarkably during the exposure. The algal growth at the 3.6 mg/L level was logarithmic, although growth inhibition was found. The algal growth at the 1.3, 0.46 and 0.058 mg/L levels was close to that in the control, although growth inhibition was found. The algal growth at the 0.16 mg/L level was the same as that in the control level. The following results of cell observation were based on the comparison with the control: Bloat cells were observed a little more at the 10 mg/L level, and slightly bloat cells were observed a little more at the 3.6 mg/L level. The conditions of cells in the other exposure levels were same as the control. In the control, the condition of cells was not abnormal. In the growth rate, statistically significant differences were observed in 3.6, 1.3, 0.46 and 0.058 mg/L levels. There was no significant difference at the 0.16 mg/L level. There was some degree of concentration-response relativity among the 0.16-3.6 mg/L levels. Inhibition rate of 0.058 mg/L level was 3.4%, which was low, and the inhibition can not be denied owing to the environmental conditions. Therefore, with the consideration of the results of cell observation, NOEC based on growth rate was estimated as 0.16 mg/L.

The environmental conditions of the test were within the suitable range; therefore, it was concluded that the study complied with the applied test guidelines.

Under the conditions of this study, the ErC50 of the test material based on the growth rate was 3.9 mg/L (95% confidence interval: 3.5-4.3 mg/L) and the NOEC was 0.16 mg/L.

Description of key information

Under the conditions of this study, the ErC50 of the test material based on the growth rate was 3.9 mg/L (95 % confidence interval: 3.5-4.3 mg/L) and the NOEC was 0.16 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

3.9 mg/L

EC10 or NOEC for freshwater algae:

0.16 mg/L

Additional information

The toxicity of the test material to algae was investigated in accordance with the standardised guideline OECD 201 and Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of the study was to determine the 0-72-hour median effective concentration (EC50) and no observed effect concentration (NOEC) by conducting an algae growth inhibition test of the test material in Pseudokirchneriella subcapitata.

The algae was exposed to the test material at nominal concentrations of 0.058, 0.16, 0.46, 1.3, 3.6 and 10 mg/L. The test material concentrations were analysed at the start and end of the test with high performance liquid chromatography. The measured concentrations of the test material in the test solution were within the range of ± 20% of nominal concentrations.

ErC50 of the test material based on the growth rate was found to be 3.9 mg/L (95% confidence interval: 3.5-4.3 mg/L). At the 10 mg/L level, algal growth was inhibited remarkably during the exposure. The algal growth at the 3.6 mg/L level was logarithmic, although growth inhibition was found. The algal growth at the 1.3, 0.46 and 0.058 mg/L levels was close to that in the control, although growth inhibition was found. The algal growth at the 0.16 mg/L level was the same as that in the control level. The following results of cell observation were based on the comparison with the control: Bloat cells were observed a little more at the 10 mg/L level, and slightly bloat cells were observed a little more at the 3.6 mg/L level. The conditions of cells in the other exposure levels were same as the control. In the control, the condition of cells was not abnormal. In the growth rate, statistically significant differences were observed in 3.6, 1.3, 0.46 and 0.058 mg/L levels. There was no significant difference at the 0.16 mg/L level. There was some degree of concentration-response relativity among the 0.16-3.6 mg/L levels. Inhibition rate of 0.058 mg/L level was 3.4%, which was low, and the inhibition can not be denied owing to the environmental conditions. Therefore, with the consideration of the results of cell observation, NOEC based on growth rate was estimated as 0.16 mg/L.

The environmental conditions of the test were within the suitable range; therefore, it was concluded that the study complied with the applied test guidelines.

Under the conditions of this study, the ErC50 of the test material based on the growth rate was 3.9 mg/L (95% confidence interval: 3.5-4.3 mg/L) and the NOEC was 0.16 mg/L.