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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 April 2009 - 11 June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material:
PHYSICO-CHEMICAL PROPERTIES
- Density at 20°C: 840 kg/m3
- Water solubility (under test conditions): very soluble
- Name of test material (as cited in study report): 3-isopropoxypropylamine
- Physical state: liquid
- Analytical purity: 99.86%
- Lot/batch No.: A1V4KQ010101
- Expiration date of the lot/batch: 27/01/2011
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
preliminary test: control, 1.00, 4.00, 12.50, 25.00, 50.00, 100.00 mg/L
definitive test: control, 25.00, 33.00, 43.40, 57.40, 75.80, 100.00 mg/L
Vehicle:
no
Details on test solutions:
Osmotically filtered water was used to prepare the dilution water. Chemical reagents used for the preparation of dilution water were of "analytical grade".
As 3-isopropoxypropylamine is soluble in water, solutions were prepared on the starting day of the test. For the range finding test, a stock solution at 100 mg/L was prepared 1 hour before the beginning of the test by mixing 100 mg of the test item in 1 litre of dilution water. It was used to prepare the convenient range of concentrations: 100, 50, 25, 12.5, 4 and 1 mg/L.

For the definitive test a stock solution at 100 mg/L solution was prepared the day of the test and used to prepare the convenient range of concentrations: 100, 75.8, 57.4, 43.4, 33 and 25 mg/L.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: freshwater alga
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (Ambleside, UK)
- Method of cultivation: Two flasks, each containing approximately 100 mL of axenic stock culture of algae are incubated at 23 ± 1 °C under lighting (photoperiod: 16 hours of illumination, 8 hours of darkness), slowly continuously shaken. These stock cultures are renewed every week, using two new cultures.

ACCLIMATION
Three days before the beginning of the study two pre-cultures were prepared by inoculating each stock suspension of algae (5 mL) into sterile dilution water (500 mL). The pre-cultures were incubated under the sampe conditions as those used for the stock cultures. Only one of the two pre-cultures was used to inoculate the test flasks; the second one was to be used only if the first one was damaged.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23 ± 1°C
pH:
Labelled Bl blank: non-inoculated blank containing only dilution water without test item
Labelled T blank: inoculated control flask

At initiation of the test: 7.96 (blank control), 9.03 (25.0 mg/L), 9.25 (33.0 mg/L), 9.37 (43.4 mg/L), 9.51 (57.4 mg/L), 9.67 (75.8 mg/L), 9.89 (100 mg/L), adjusted pH of 8.28 (at 100 mg/L)
After 72 hours exposure: 7.97 (blank control), 9.81 (inoculated control), 9.13 (25.0 mg/L), 9.12 (33.0 mg/L), 8.52 (43.4 mg/L), 8.34 (57.4 mg/L), 8.30 (75.8 mg/L), 8.36 (100 mg/L), adjusted pH of 8.66 (at 100 mg/L)
Dissolved oxygen:
At initiation of test: 8.9 mg/L (control Bl, 25.0, 33.0, 43.4, 57.4, 75.8 mg/L), 9.0 mg/L (100 mg/L)
After 72 hours exposure: 9.1 mg/L (control Bl, 75.8 mg/L, 100 mg/L), 12.1 mg/L (control T), 11.2 mg/L (25.0 mg/L), 11.1 mg/L (33.0 mg/L), 9.8 mg/L (43.4 and 57.4 mg/L)
Salinity:
Not applicable
Nominal and measured concentrations:
nominal concentrations (preliminary test): 100, 50, 12.5, 4, 1 mg/L
nominal concentrations (definitive test): 100, 75.8, 57.4, 43.4, 33 and 25 mg/L
measured concentrations (definitive test - measurements in non inoculated solutions):
initial: 109.9, 83.25, 67.33, 48.53, 37.86, 27.45 mg/L, < LOD (control Bl)
final: 98.32, 77.72, 60.06, 43.24, 33.68, 24.13, < LOD (control Bl)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass bottles stoppered with cellulose bungs
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 120 mL, 50 mL filling volume
- Aeration: no
- Initial cells density: density of pre-culture at start of the preliminary test: 1.750 x 1E06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Flasks were constantly shaken with a rotation at 20 rpm

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Photoperiod: constantly illuminated
- Light intensity and quality: between 6000 and 10000 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorimeter: Fluoresence was then determined using a cytofluorimeter and by comparison with a calibration range, cell density was determined, according to the measured fluorescence (at 24, 48, and 72 hours)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.32 for definitive test
- Justification for using less concentrations than requested by guideline:
- Range finding study: concentrations tested: 100, 50, 25, 12.5, 4, 1 mg/L
- Results used to determine the conditions for the definitive study: percentage inhibition of growth rate in preliminary test: 1.78% (1 mg/L), 1.20% (4 mg/L), 0.39% (12.5 mg/L), 2.16% (25 mg/L), 25.07% (50 mg/L), 100.00% (100 mg/L)
Reference substance (positive control):
yes
Remarks:
sodium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 28 - 75 mg/L
Remarks:
test solutions without pH adjustment
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CI: 19 - 38 mg/L
Remarks:
test solutions without pH adjustment
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CI: 6.6 - 40 mg/L
Remarks:
test solutions without pH adjustment
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: based on absence of effects in test solutions with adjusted pH
Details on results:
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless over the period of the test. No precipitation was observed at the end of the test.
Microscopic observation confirmed that the algae appeared normal at the end of the test: the normal shape of P. subcapitata algae is a crescent shaped cell with an average length of 5 - 10 µm.

The test results refer to an unneutralised test item. 3-isopropoxypropylamine is no longer toxic after neutralisation.
Results with reference substance (positive control):
The sensitivity of the test system and the methodology are evaluated every two months by performing an algal growth inhibition test on sodium dichromate. The nearest value of ErC50 and EbC50 obtained on June 05, 2009 were respectively 0.98 mg/L and 0.46 mg/L.
ISO 8692 reports the following results for an inter-laboratory exercise on sodium dichromate:
ErC50: 0.60 to 1.03 mg/L
EbC50: 0.20 to 0.75 mg/L
Reported statistics and error estimates:
The growth inhibition data are analysed using an Excel program. It was designed to calculate the EC50 value and the 95% confidence interval. Probit analysis is generally used to calculate the 24, 48 and 72 hour EC50 values.

The final concentrations of 3-isopropoxypropylamine were maintained within the designated limit of 80% of the initial concentration in non-inoculated flasks. Thus, the test item concentration was maintained throughout the duration of the test.

Since the final measured concentrations in non-inoculated flasks were found to be equivalent to at least 80% of the nominal concentration, estimation of effective concentrations (EC50) were based on nominal values.

The study was performed in compliance with the following quality criteria:

- The biomass in the control cultures has increased exponentially by a factor 16 within the 72-hour test period. This corresponds to a specific growth rate of 0.92/day.

- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35%. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures.

- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7%.

Validity criteria fulfilled:
yes
Conclusions:
A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance 3-isopropoxypropylamine according to the OECD guideline 201 (GLP conditions). The 72-hour EC10 and EC50 for growth rate (calculated on the basis of nominal concentrations) in test solutions in which pH was not adjusted were 29 and 44 mg/L, respectively. The pH was very high (> 9) in a concentration dependent matter in all test solutions. Although the OECD guideline 201 does not specify a recommended pH range for the test species, algal growth was also observed in a test solution at a nominal concentration of 100 mg/L in which pH was lowered to an environmentally relevant level. In this test solution, no significant growth inhibition was observed. Therefore, the observed effect was considered related to the pH increasing effect of the test substance.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Data from the related substance 3-isopropoxypropylamine are used to cover this endpoint. The justification for read across is attached to IUCLID Section 13.
Reason / purpose:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
44 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Experimental data on 3-isopropoxypropylamine (obtained in test solutions without pH adjustment) used for read across.
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
29 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Experimental data on 3-isopropoxypropylamine (obtained in test solutions without pH adjustment) used for read across.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Experimental data on 3-isopropoxypropylamine (obtained in test solutions in which pH was adjusted) supporting the read across strategy.

Description of key information

Read across is performed using a study with the structural analogue 3-isopropoxypropylamine, performed according to OECD guideline 201. This study was considered as the key study for endpoint coverage, yielding an EC50(72h) value of 44 mg/L and an EC10(72h) value of 29 mg/L based on growth rate. It should be noted that these values should be considered as worst case values, as no significant growth inhibition was observed in a test solution of 100 mg/L in which the pH was lowered to environmentally relevant levels (i.e. EC50(72h) > 100 mg/L). The observed growth inhibition in the test solutions in which pH was not adjusted was therefore considered as a pH effect, and no evidence for a direct intrinsic toxic effect of the test substance was obtained. The read across justification document is attached to IUCLID Section 13.

Key value for chemical safety assessment

EC50 for freshwater algae:
44 mg/L

Additional information