Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)

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:

20 July 2018 to 23 August 2018

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

Analytical monitoring:

yes

Details on sampling:

- Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the glass wool containing the undissolved residue was kept for possible analysis.
- Frequency: at t=0 h, t=24 h and t=72 h.
- Volume: 2.0 mL from the approximate centre of the test vessels.
- Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.
- At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at a WAF prepared at a loading rate of 32 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The batch of test material tested was an amber liquid UVCB substance which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test material.
- Preparation of test solutions started with loading rates individually prepared ranging between 1.0 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test material in medium. The obtained mixtures were allowed to settle for approximately one day. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations. At the end of the preparation procedure, WAFs prepared at loading rates up to and including 10 mg/L were clear and colourless, while WAFs prepared at loading rates > 10 mg/L were yellow. Additionally, the highest test concentration in both tests was observed with a laser pen to check for the presence of Tyndall effect. Undissolved particles were observed. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Any residual volumes were discarded.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source: In-house laboratory culture.
- Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

ALGAE CULTURE
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24 °C.
- Light intensity: 60 to 120 μE/m²/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap water purified by reverse osmosis) with the following composition: NaNO3 500 mg/L, K2HPO4 39.5 mg/L, MgSO4.7H2O 75 mg/L, Na2CO3 20 mg/L, C6H8O7.H2O 6 mg/L, NH4NO3 330 mg/L, CaCl2.2H2O 35 mg/L, C6H5FeO7.xH2O 6 mg/L, H3BO3 2.9 mg/L, MnCl2.4H2O 1.81 mg/L, ZnCl2 0.11 mg/L, CuSO4.5H2O 0.08 mg/L and (NH4)6Mo7O24.4H2O 0.018 mg/L.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 μg/L, Na2EDTA.2H2O 100 μg/L, H3BO3 185 μg/L, MnCl2.4H2O 415 μg/L, ZnCl2 3 μg/L, CoCl2.6H2O 1.5 μg/L, CuCl2.2H2O 0.01 μg/L, Na2MoO4.2H2O 7 μg/L and NaHCO3 50 mg/L. Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L). pH 8.1 ± 0.2

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23 °C

pH:

6.7 - 8.1

Nominal and measured concentrations:

Nominal: 10, 18, 32, 56 and 100 mg/L WAF

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass with aluminium caps, perforated for ventilation
- Fill volume: each vessel contained 50 mL of test solution
- Initial cells density: 1 x 10^4 cells/mL
- During incubation the algal cells were kept in suspension by continuous shaking.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 1 extra replicate of each test group was performed for sampling purposes after 24 hours of exposure, and 1 or 2 replicates of each test concentration were performed without algae.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition: NH4Cl 15 mg/L, MgCl2.6H2O 12 mg/L, CaCl2.2H2O 18 mg/L, MgSO4.7H2O 15 mg/L, KH2PO4 1.6 mg/L, FeCl3.6H2O 64 μg/L, Na2EDTA.2H2O 100 μg/L, H3BO3 185 μg/L, MnCl2.4H2O 415 μg/L, ZnCl2 3 μg/L, CoCl2.6H2O 1.5 μg/L, CuCl2.2H2O 0.01 μg/L, Na2MoO4.2H2O 7 μg/L and NaHCO3 50 mg/L. Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L). pH 8.1 ± 0.2
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test, for all test concentrations. Temperature of medium was measured continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 83 to 85 μE.m^-2.s^-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Other: At the end of the final test microscopic observations were performed on the WAF prepared at loading rate of 56 mg/L and the control to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Final test concentrations: WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L.
- Combined Limit/Range-Finding Test: The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and WAF prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
* Three replicates per concentration were exposed to WAFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test.
* Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
* pH was only measured in the control and the highest test concentration.
* At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

INTERPRETATION
Acceptability of the Test
- In the control, cell density increased by an average factor of at least 16 within the exposure period (i.e. 204).
- The mean coefficient of variation for section-by-section specific growth rates in the control cultures did not exceed 35 % (i.e. 9.6 %).
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % (i.e. 0.8 %).

- Calibration Curve: Quantification of cell densities was based on a calibration curve. Cell density was plotted versus extinction using spectrophotometric measurements of a minimum of six dilutions of an algal suspension with different cell densities. The calibration curve was composed using linear regression. The software automatically calculates the cell densities based on this curve for the spectrophotometric measurements at the various points in time during the test period.

- Comparison of Average Growth Rates: The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:
µi-j = [(lnXj – lnXi) / (tj-ti)] (day^1)
Where:
μi-j = the average specific growth rate from time i to j
Xi = the biomass at time i
Xj = the biomass at time j
The average growth rate at each test material concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:
%Ir = [(µC - µT)/µC] x 100
Where: %Ir = percent inhibition in average specific growth rate
μC = mean value for average specific growth rate in the control group
μT = average specific growth rate for the treatment replicate

- Yield: The percent inhibition in yield is calculated for each treatment replicate as follows:
%Iy = [(YC – YT)/YC] x 100
Where: %Iy = percent inhibition of yield
YC = mean value for yield in the control group
YT = value for yield for the treatment replicate

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance.

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

WAF

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance.

Details on results:

COMBINED LIMIT/ RANGE-FINDING TEST
- No biologically relevant inhibition of algal growth rate or yield was found at WAFs prepared at loading rates of 1.0 and 10 mg/L, while an inhibition of 77 and 98 % for growth rate and yield, respectively, was found at the highest WAF prepared at loading rate of 100 mg/L. It should be noted that at the highest test solution, a band of test material attached to the glass was observed in all the test vessels, just above the surface of the test solution, throughout the test. This was supposedly caused by oversaturation.
- Samples taken from all the test solutions were analysed. No clear test material related responses could be measured throughout the test, with the exception of the measured concentration at the highest WAF at the start of the test, which was 0.91 mg/L. It should be noted that responses were observed in the analytical blanks in all three days of analysis, most probably caused by carry over.
- All test conditions were maintained within the limits prescribed by the study plan.

MAIN TEST
- Measured Test Material Concentrations: Samples taken from all test concentrations and the control were analysed. At the start of the test, a minimal increase in measured concentrations was observed in WAFs prepared at loading rate of 18 mg/L and higher, i.e. from 0.012 to 0.017 mg/L, indicating proper preparation of WAFs. These concentrations decreased below the limit of detection at the end of the test. At the lowest test concentration, i.e. a WAF prepared at a loading rate of 10 mg/L, the measured concentration was below the limit of detection already at the start of the test. It should be noted that, except the lowest test solution, a band of test material attached to the glass was observed in all the test vessels, just above the surface of the test solution, throughout the test. This was supposedly caused by oversaturation, since initially analysed concentrations were also higher that the calculated water solubility. As the test material was a UVCB substance and WAFs were prepared, the effect parameters were expressed based nominal loading rates.
- Inhibition of Growth Rate and Inhibition of Yield: Increasing inhibition of both growth rate and yield (up to 45 and 91 % when compared to the control group, respectively) was observed with increasing loading rates at the end of the test. The effects observed at all loading rates were statistically significant. However, the effects observed at the three lowest loading rates for growth rate inhibition were considered biologically not relevant (i.e. were <10 %). It should be noted that effects observed at loading rates of 18 mg/L and higher might be overestimated as oversaturation was observed in these treatments. Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF prepared at a loading rate of 56 mg/L when compared to the control.

Results with reference substance (positive control):

- Potassium dichromate inhibited growth rate and yield of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
- The EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95 % confidence interval ranging from 0.88 to 0.93 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ERC50 for the algal culture tested corresponds with this range.
- The EC50 for yield inhibition (72h-EYC50) was 0.30 mg/L with a 95 % confidence interval ranging from 0.29 to 0.31 mg/L. The historical ranges for yield inhibition lie between 0.43 and 1.1 mg/L.

Reported statistics and error estimates:

For determination of the NOEL and the ELx the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
Calculation of ELx-values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal loading rate of the test material.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Summary of Results of the Total Test Period

Loading Rate (mg/L)	% Inhibition of Growth Rate	% Inhibition of Yield
Control	-	-
10	5.3#	25*
18	6.6#	30*
32	8.4#	36*
56	44*	91*
100	45*	91*

# - effect was statistically significant however biologically not relevant (<10%), *- effect was statistically significant

 

Table 2: Effect Parameters

Parameter (mg/L)		NOEL*	NOEL#	EL10	EL20	EL50
Growth Rate	Value	<10	32	21	36	>100
	Lower 95%-CL			20	35
	Upper 95%-CL			22	38
Yield	Value	<10	<10	8.7	13	29
	Lower 95%-CL			8.3	13	28
	Upper 95%-CL			9.2	14	30

* - based on statistical significance, # - based on biological relevance.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study, the test material inhibited growth rate and yield of the fresh water algae species significantly at a loading rates of 10 mg/L and above. The EL50 for growth rate inhibition (72h-ERL50) exceeded a loading rate of 100 mg/L, at which the initially measured concentration was higher than the calculated water solubility of the test material. The EL50 for yield inhibition (72h-EYL50) was 29 mg/L with 95 % confidence intervals between 28 and 30 mg/L. The 72h-NOEL for growth rate inhibition was below the loading rate of 10 mg/L, based on statistical significance. The 72h-NOEL for growth rate inhibition was 32 mg/L, based on biological relevance. The 72h-NOEL for yield inhibition was below the loading rate of 10 mg/L, based on statistical significance and biological relevance.

Executive summary:

The toxicity of the test material to aquatic algae was determined in accordance with the standardised guideline OECD 201, under GLP conditions.

The objective of the study was to evaluate the test material for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEL, EL10, EL20 and EL50 for both inhibition of growth rate and inhibition of yield.

Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. At the start of the test, a minimal increase in measured concentrations was observed in WAFs prepared at loading rate of 18 mg/L and above, i.e. from 0.012 to 0.017 mg/L, indicating proper preparation of WAFs. These concentration decreased below the limit of detection at the end of the test. At the lowest test concentration, i.e. a WAF prepared at a loading rate of 10 mg/L, the measured concentration was below the limit of detection already at the start of the test. As the test material is a UVCB substance, effect parameters were based on the nominal loading rates. Increasing inhibition of both growth rate and yield was observed with increasing loading rates when compared to the control group, up to 45 and 91%, respectively, at the end of the test.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of the present study, the test material inhibited growth rate and yield of the fresh water algae species significantly at a loading rates of 10 mg/L and above. The EL50 for growth rate inhibition (72h-ERL50) exceeded a loading rate of 100 mg/L, at which the initially measured concentration was higher than the calculated water solubility of the test material. The EL50 for yield inhibition (72h-EYL50) was 29 mg/L with 95 % confidence intervals between 28 and 30 mg/L. The 72h-NOEL for growth rate inhibition was below the loading rate of 10 mg/L, based on statistical significance. The 72h-NOEL for growth rate inhibition was 32 mg/L, based on biological relevance. The 72h-NOEL for yield inhibition was below the loading rate of 10 mg/L, based on statistical significance and biological relevance.

Description of key information

In conclusion, under the conditions of the study, the test material inhibited growth rate and yield of the fresh water algae species significantly at a loading rates of 10 mg/L and above. The EL50 for growth rate inhibition (72h-ERL50) exceeded a loading rate of 100 mg/L, at which the initially measured concentration was higher than the calculated water solubility of the test material. The EL50 for yield inhibition (72h-EYL50) was 29 mg/L with 95 % confidence intervals between 28 and 30 mg/L. The 72h-NOEL for growth rate inhibition was below the loading rate of 10 mg/L, based on statistical significance. The 72h-NOEL for growth rate inhibition was 32 mg/L, based on biological relevance. The 72h-NOEL for yield inhibition was below the loading rate of 10 mg/L, based on statistical significance and biological relevance.

Additional information

The toxicity of the test material to aquatic algae was determined in accordance with the standardised guideline OECD 201, 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 evaluate the test material for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOEL, EL10, EL20 and EL50 for both inhibition of growth rate and inhibition of yield.

Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. A final test was performed based on the results of a preceding combined limit/range-finding test. Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs individually prepared at loading rates of 10, 18, 32, 56 and 100 mg/L. The initial algal cell density was 10^4 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from all test concentrations and the control were analysed. At the start of the test, a minimal increase in measured concentrations was observed in WAFs prepared at loading rate of 18 mg/L and above, i.e. from 0.012 to 0.017 mg/L, indicating proper preparation of WAFs. These concentration decreased below the limit of detection at the end of the test. At the lowest test concentration, i.e. a WAF prepared at a loading rate of 10 mg/L, the measured concentration was below the limit of detection already at the start of the test. As the test material is a UVCB substance, effect parameters were based on the nominal loading rates. Increasing inhibition of both growth rate and yield was observed with increasing loading rates when compared to the control group, up to 45 and 91%, respectively, at the end of the test.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, under the conditions of the study, the test material inhibited growth rate and yield of the fresh water algae species significantly at a loading rates of 10 mg/L and above. The EL50 for growth rate inhibition (72h-ERL50) exceeded a loading rate of 100 mg/L, at which the initially measured concentration was higher than the calculated water solubility of the test material. The EL50 for yield inhibition (72h-EYL50) was 29 mg/L with 95 % confidence intervals between 28 and 30 mg/L. The 72h-NOEL for growth rate inhibition was below the loading rate of 10 mg/L, based on statistical significance. The 72h-NOEL for growth rate inhibition was 32 mg/L, based on biological relevance. The 72h-NOEL for yield inhibition was below the loading rate of 10 mg/L, based on statistical significance and biological relevance.