(2S-cis)-(diphenylmethyl)-N-(phenylmethyl)-1-azabicyclo[2.2.2.]octan-3-amine (1-R camphor-10-sulfonate)

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-810-6

EC Name:

-

Molecular formula:

C27 H30 N2 * C10 H16 O4 S

IUPAC Name:

(2S-cis)-(diphenylmethyl)-N-(phenylmethyl)-1-azabicyclo[2.2.2.]octan-3-amine (1-R camphor-10-sulfonate)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: CP-163,625-BV
Batch (Lot) Number: INT 18 320
Expiry date: 16 April 2020 (retest date)
Physical Description: White powder
Purity/Composition: 97.8
Storage Conditions: At room temperature

Sampling and analysis

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 filter containing the undissolved residue was kept for possible analysis.
Frequency at t=0 h and t=72 h.
Volume 2.0 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
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 1.0% of the SS but without algae and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of CP-163,625-BV tested was a white powder with a purity of 97.8% which was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a 15 minute period of ultrasonic treatment followed by three days of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
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 104 cells/mL.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test System
Species Raphidocelis subcapitata, 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.

Freshwater 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; Millipore Corp., Bedford, Mass., USA) 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
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture
Three 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 104 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
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).

pH:

The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).

Dissolved oxygen:

N/A

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal: 0 ,0.1, 0.32, 1, 3.2, 10 , 32, 100 mg/L
Measured: 0, 0.06, 0.2, 0.62, 2.0, 6.0, 20, 62 mg/L

Details on test conditions:

Testing Strategy and Experimental Design
Combined Limit/Range-Finding Test
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to an untreated control and a SS 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 solutions containing 1.0 and 10% of the SS in the combined range-finding test.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Lower concentrations were measured only at the end of the exposure period.
• One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.
• 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.
• Samples for possible analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Final Test
Test Concentrations
CP-163,625-BV Solutions containing 0.10, 0.32, 1.0, 3.2 and 10% of the SS, prepared at a loading rate of 100 mg/L.
Control Test medium without test item or other additives.
Replicates 3 replicates of each test concentration, 6 replicates of the control,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

Test Procedure and Conditions
Test duration 72 hours
Test type Static
Test vessels 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution.
Test Medium M2
Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity within the range of 62 to 65 µE.m-2.s-1.
Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.9 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.44 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

cell number

Results with reference substance (positive control):

The study met the acceptability criteria prescribed by the study plan and was considered valid.
Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 1.30 mg/L with a 95% confidence interval ranging from 1.29 to 1.32 mg/L.
The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.
In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with ISO International Standard 8692, February 2012 and the historical data collected at Charles River Den Bosch.

Any other information on results incl. tables

Combined Limit/Range-Finding Test

The mean cell densities measured during the combined limit/range-finding test are presented inTable 1.Table 2andTable 3present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

After 72 hours of exposure, growth rate and yield were inhibited by 13 and 52%, respectively, at the lowest concentration tested. In the solution containing 10% of the SS, 100% inhibition of growth rate and yield was found. Recorded effects were slightly lower at the highest test concentration, i.e. 87% growth rate and 99% yield inhibition.

Based on these results, samples taken from all test concentrations were analysed. The measured concentrations at the start of the test were 0.59, 5.9 and 59 mg/L, respectively. The concentrations remained stable throughout the test, i.e. were in the range of 96 to 107% relative to the initial concentrations at the end of the test (see also Table 2 of the appended Analytical Report).

All test conditions were maintained within the limits prescribed by the study plan.

Table 1          
Mean Cell Densities (x10⁴Cells/mL) during the Combined Limit/Range-Finding Test

Time (h)	CP-163,625-BV %SS prep. at a loading rate of 100 mg/L
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	12	n.d.	n.d.	1.0
48	72	n.d.	n.d.	1.2
72	340	163	1.0	2.9

n.d. – not determined.

Table 2          
Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

CP-163,625-BV %SS prep. at a loading rate of 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	1.942	0.0136	6
1.0	1.698	0.0298	3	13
10	0.000	0.0000	3	100
100	0.248	0.2790	6	87

Table 3          
Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

CP-163,625-BV %SS prep. at a loading rate of 100 mg/L	Mean	Std. Dev.	n	%Inhibition
Control	338.5	14.02	6
1.0	162.4	14.81	3	52
10	0.0	0.00	3	100
100	1.9	2.55	6	99

 Final Test

 Measured Test Item Concentrations

Samples taken from all test concentrations and the control were analysed. The measured concentrations at the start of the test were 0.060, 0.20, 0.62, 2.0 and 6.0 mg/L in solutions containing 0.10, 0.32, 1.0, 3.2 and 10% of the SS prepared at a loading rate of 100 mg/L, respectively. The concentrations remained stable throughout the test, i.e. were in the range of 97 to 106% relative to the initial concentrations at the end of the test. In view of these results, effect parameters were expressed based on the initially measured concentrations.

It should be noted that small test item responses were detected in the control samples. The concentration measured in the sample taken at the start of the test could be allocated to carry-over since a similar response was present in the blank Quality Control (QC) samples and analytical blanks. The test item concentration measured in the control sample taken at the end of the test was considerably higher and therefore must have originated from a different source. Nevertheless, it was considered that no test item was present in the exposure vessels during the test as no response with such order of magnitude was measured at the start of the test, and therefore, was most likely introduced during sampling procedures. Therefore, it was considered that this concentration had no effect on the effect parameters derived in this study.

The concentrations measured in the samples taken from the solution incubated without algae was comparable to the concentrations analysed in its counterpart incubated with algae. Hence, it can be stated that the presence of the algae had no effect on the concentration of the test item in test medium throughout the test.

Mean Cell Densities

The individual and group mean cell densities measured at 24h intervals are given in Table 9.

Inhibition of Growth Rate and Inhibition of Yield

Table 4shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereasTable 5shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized inTable 6(see Appendix 1 for the individual values). Statistical analysis of the data is shown inAppendix 2andAppendix 3.

Statistically significant growth rate and yield inhibition was found at concentrations of 0.62 mg/L and higher. At the end of the test, 25% growth rate and 75% yield inhibition was found at a concentration of 0.62 mg/L, while 100% growth rate and yield inhibition was recorded at concentrations of 2.0 and 6.0 mg/L. No significant differences were found for growth rate or yield between the control and the two lowest concentrations tested. The NOEC was thus 0.20 mg/L for both growth rate and yield inhibition.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.62 mg/L when compared to the control.

Table 4          
Growth Rate and Percentage Inhibition for the Total Test Period

CP-163,625-BV Measured conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.863	0.0185	6
0.060	1.859	0.0265	3	0.20
0.20	1.849	0.0337	3	0.76
0.62	1.404	0.0343	3	25*
2.0	0.000	0.0000	3	100*
6.0	0.000	0.0000	3	100*

* Effect was statistically significant.

Table 5          
Growth Rate and Percentage Inhibition at Different Time Intervals

 

CP-163,625-BV Measured conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.362		1.747		1.481
0.060	3	2.374	-0.53	1.739	0.44	1.464	1.1
0.20	3	2.420	-2.5	1.613	7.7	1.514	-2.2
0.62	3	2.161	8.5	0.582	67	1.468	0.82
2.0	3	1.087	54	-0.205	112	-0.882	160
6.0	3	0.092	96	-0.092	105	0.000	100

Table 6          
Yield and Percentage Inhibition for the Total Test Period

CP-163,625-BV Measured conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	266.8	14.47	6
0.060	264.1	20.97	3	1.0
0.20	256.2	25.70	3	4.0
0.62	66.7	6.89	3	75*
2.0	0.0	0.00	3	100*
6.0	0.0	0.00	3	100*

* Effect was statistically significant.

 

Determination of Effect Concentrations

Table 7 shows the effect parameters based on measured concentrations.

Table 7          
Effect Parameters

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	0.20	0.41	0.56	0.90
	lower 95%-cl		0.40	0.54	0.87
	upper 95%-cl		0.43	0.58	0.92
Yield	Value	0.20	0.23	0.29	0.44
	lower 95%-cl		0.19	0.25	0.40
	upper 95%-cl		0.27	0.33	0.48

cl – confidence limit.

Experimental Conditions

Table 8 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21‑24°C, constant within ±1°C).

Table 8          
pH Levels Recorded during the Final Test

CP-163,625-BV Measured conc. (mg/L)	pH
	t=0h	t=72h
Control	8.3	8.3
0.060	8.3	8.2
0.20	8.3	8.2
0.62	8.2	8.1
2.0	8.2	8.0
6.0	8.2	8.0

  Table 9          
Individual Cell Densities (x10⁴cells/mL)

Time	Replicate	CP-163,625-BV; Measured conc. (mg/L)
		Control	0.060	0.20	0.62	2.0	6.0
0 h	1 2 3 4 5 6	1.000	1.000	1.000	1.000	1.000	1.000
	2	1.000	1.000	1.000	1.000	1.000	1.000
	3	1.000	1.000	1.000	1.000	1.000	1.000
	4	1.000
	5	1.000
	6	1.000
n:		6	3	3	3	3	3
Mean:		1.0	1.0	1.0	1.0	1.0	1.0
Std.Dev.:		0.0	0.0	0.0	0.0	0.0	0.0
CV:		0.0	0.0	0.0	0.0	0.0	0.0
24 h	1 2 3 4 5 6	10.845	10.535	12.008	9.423	2.557	1.208
	2	11.123	10.845	10.540	8.776	3.615	1.000
	3	10.636	10.850	11.242	7.914	2.822	1.090
	4	9.646
	5	10.786
	6	10.677
n:		6	3	3	3	3	3
Mean:		10.6	10.7	11.3	8.7	3.0	1.1
Std.Dev.:		0.5	0.2	0.7	0.8	0.6	0.1
CV:		4.8	1.7	6.5	8.7	18.4	9.5
48 h	1 2 3 4 5 6	64.739	59.706	61.137	15.810	1.988	1.000
	2	69.448	61.292	54.564	14.884	2.690	1.000
	3	62.824	62.500	53.880	15.933	2.640	1.000
	4	53.447
	5	56.857
	6	59.182
n:		6	3	3	3	3	3
Mean:		61.1	61.2	56.5	15.5	2.4	1.0
Std.Dev.:		5.8	1.4	4.0	0.6	0.4	0.0
CV:		9.4	2.3	7.1	3.7	16.1	0.0
72 h	1 2 3 4 5 6	271.223	243.437	281.416	74.312	1.000	1.000
	2	283.855	266.427	260.067	68.231	1.000	1.000
	3	276.420	285.309	230.253	60.554	1.000	1.000
	4	241.568
	5	264.307
	6	269.563
n:		6	3	3	3	3	3
Mean:		267.8	265.1	257.2	67.7	1.0	1.0
Std.Dev.:		14.5	21.0	25.7	6.9	0.0	0.0
CV:		5.4	7.9	10.0	10.2	0.0	0.0

According to OECD 201, the factor of the biomass parameter, measured in the control between 0 and 72 h, must be at least 16. In the current test it was found to be 268. The test fulfils this validity criterion.

Table 10        
Individual Growth Rates (day^-1)

Time	Replicate	CP-163,625-BV; Measured conc. (mg/L)
		Control	0.060	0.20	0.62	2.0	6.0
0-24 h	1 2 3 4 5 6	2.384	2.355	2.486	2.243	0.939	0.189
	2	2.409	2.384	2.355	2.172	1.285	0.000
	3	2.364	2.384	2.420	2.069	1.037	0.086
	4	2.267
	5	2.378
	6	2.368
Mean:		2.362	2.374	2.420	2.161	1.087	0.092
Std.Dev.:		0.0492	0.0169	0.0652	0.0878	0.1784	0.0946
n:		6	3	3	3	3	3
CV:		2.083	0.711	2.694	4.06	16.41	103.151
0-48 h	1 2 3 4 5 6	2.085	2.045	2.057	1.380	0.344	0.000
	2	2.120	2.058	2.000	1.350	0.495	0.000
	3	2.070	2.068	1.993	1.384	0.485	0.000
	4	1.989
	5	2.020
	6	2.040
Mean:		2.054	2.057	2.017	1.372	0.441	0.000
Std.Dev.:		0.0472	0.0115	0.0348	0.0186	0.0847	0.000
n:		6	3	3	3	3	3
CV:		2.297	0.558	1.726	1.359	19.201	n.a.
0-72 h	1 2 3 4 5 6	1.868	1.832	1.880	1.436	0.000	0.000
	2	1.883	1.862	1.854	1.408	0.000	0.000
	3	1.874	1.885	1.813	1.368	0.000	0.000
	4	1.829
	5	1.859
	6	1.866
Mean:		1.863	1.859	1.849	1.404	0.000	0.000
Std.Dev.:		0.0185	0.0265	0.0337	0.0343	0.000	0.000
n:		6	3	3	3	3	3
CV:		0.993	1.427	1.823	2.442	n.a.	n.a.

n.a. – not applicable.

The coefficient of variation of the mean specific growth rate replicates in the control between 0 and 72 h was: 0.99%. According to OECD 201, the coefficient of variation of the mean specific growth rate, measured in the control from 0 to 72 h, must not exceed 7%.The test fulfils this validity criterion.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, CP 163,625-BV inhibited growth rate and yield of this freshwater algae species significantly at measured concentrations of 0.62 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.87 to 0.92 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 0.44 mg/L with a 95% confidence interval ranging from 0.40 to 0.48 mg/L.
The 72h-NOEC for both growth rate and yield inhibition was 0.20 mg/L.
 

Executive summary:

The objectiveofthe study was to evaluate CP-163,625-BV for its ability to generate toxic effects inRaphidocelis subcapitataduring an exposure period of 72 hours and, if possible, to determine the NOEC, EC₁₀, EC₂₀and EC₅₀for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23,2019.

The batch of CP-163,625-BV tested was a white powder with a purity of 97.8% which was not completely soluble in test medium at the loading rate initially prepared.A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

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 solutions containing 0.10, 0.32, 1.0, 3.2 and 10% of the SS prepared at a loading rate of 100 mg/L. The initial algal cell density was 10⁴cells/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. The measured concentrationsat the start of the testwere 0.060, 0.20, 0.62, 2.0 and 6.0 mg/L in solutions containing 0.10, 0.32, 1.0, 3.2 and 10% of the SS prepared at a loading rate of 100 mg/L, respectively. The concentrations remained stable throughout the test, i.e. were in the range of 97 to 106% relative to the initial concentrations at the end of the test. In view of these results, effect parameters were expressed as measured concentrations.

Statistically significant growth rate and yield inhibition was found at concentrations of 0.62 mg/L and higher. At the end of the test, 25% growth rate and 75% yield inhibition was found at a concentration of 0.62 mg/L, while 100% growth rate and yield inhibition was recorded at concentrations of 2.0 and 6.0 mg/L. No significant differences were found for growth rate or yield between the control and the two lowest concentrations tested.

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

The effect parameters obtained in this study are summarized in the table below.

Parameter (mg/L)		NOEC	EC10	EC20	EC50
Growth rate	Value	0.20	0.41	0.56	0.90
	lower 95%-cl		0.40	0.54	0.87
	upper 95%-cl		0.43	0.58	0.92
Yield	Value	0.20	0.23	0.29	0.44
	lower 95%-cl		0.19	0.25	0.40
	upper 95%-cl		0.27	0.33	0.48

cl – confidence limit.