Niels Nyholm

Primary biodegradation of veterinary antibiotics in aerobic and anaerobic surface water simulation systems

The primary aerobic and anaerobic biodegradability at intermediate concentrations (50-5000 microg/l) of the antibiotics olaquindox (OLA), metronidazole (MET), tylosin (TYL) and oxytetracycline (OTC) was studied in a simple shake flask system simulating the conditions in surface waters. The purpose of the study was to provide rate data for primary biodegradation in the scenario where antibiotics pollute surface waters as a result of run-off from arable land. The source of antibiotics may be application of manure as fertilizer or excreta of grazing animals. Assuming first-order degradation kinetics, ranges of half-lives for aerobic degradation of the four antibiotics studied were 4-8 days (OLA), 9.5-40 days (TYL), 14-104 days (MET) and 42-46 days (OTC). OLA and OTC were degraded with no initial lag phase whereas lag phases from 2 to 34 days (MET) and 31 to 40 days (TYL) were observed for other substances. The biodegradation behaviour was influenced by neither the concentrations of antibiotics nor the time of the year and location for sampling of surface water. Addition of 1 g/l of sediment or 3 mg/l of activated sludge from wastewater treatment increased the biodegradation potential which is believed to be the result of increased bacterial concentration in the test solution. Biodegradation was significantly slower in tests conducted in absence of oxygen. Assessments of the toxic properties of antibiotics by studying the influence on the biodegradation rates of 14C-aniline at different concentrations of antibiotics showed that no tests were conducted at toxic concentrations.

A simple in vitro fluorescence method for biomass measurements in algal growth inhibition tests

Abstract The estimation of biomass concentrations in algal growth inhibition tests from measurements of pigment fluorescence in extracts of 20% sample (final v/v) prepared by direct addition to dimethylsulfoxide/acetone solvent offers several advantages compared to currently used direct or indirect methods. The extraction stops the electron transfer and other processes which interact with chlorophyll fluorescence when measured in vivo. As a result the response is stabilized and the sensitivity improved. The injection method is very fast, has a high potential for automation, allows storage of samples and is suitable for small sample volumes (e.g., 0.2 ml). The typical initial cell density in standard toxicity tests of 104 cells ml−1 of Selenastrum capricornutum was measured precisely with a standard fluorimeter set-up, and 103 cells ml−1 of S. capricornutum was measured reliably with a sensitive fluorimeter. At low levels of toxicity by the model test compound potassium dichromate, the proposed fluorescence method resulted in very similar inhibition figures as obtained with electronic particle counting. At high levels of toxicity, on the other hand, biomass determinations from pigment fluorescence readings were markedly affected by toxicant-induced changes of the algal physiology. The low effect part of a dose response curve is normally that one of major interest, and biomass estimation errors associated with fluorescence measurements on extracts are thus considered acceptable in most situations. When the entire dataset was applied for endpoint estimation by the Weibull model, EC-1 estimates were markedly affected by the curve fitting to data in the high inhibition range, while EC-10 and EC-20 were less and EC-50 almost unaffected. The method is expected to be less suitable for toxicity testing of herbicides specifically inhibiting photosynthesis.

Response variable in algal growth inhibition tests—Biomass or growth rate?

Abstract A number of response variables can be derived from growth inhibition toxicity experiments with microorganisms; currently, there is a controversy regarding which variable to specify in standardized test protocols with algae. Yield or biomass at a specified time is the variable used most frequently. Specific average growth rate is a debated alternative, and appreciable differences may exist between EC figures derived from the same set of data using either test endpoint. The problem is reviewed and analysed mathematically. It is concluded that from a theoretical point of view, growth rate is a better response variable than biomass, e.g. because EC figures estimated from growth rates are less dependent on particular test system parameters. However, as the use of both methods to analyse data will probably continue, it is important to be aware of the potential differences between estimated EC figures.

A Miniscale Algal Toxicity Test

Abstract A simple miniscale (approx. 1–2.5 ml) toxicity test procedure with the freshwater green alga Selenastrum capricornutum is described. The procedure fulfils the validity criteria of the ISO (International Association for Standardization) standard test protocol. Practically identical concentration-response curves were obtained with the ISO standard test and the minitest for potassium dichromate and 3,5-dichlorophenol. The minitest is conveniently carried out using 2.5 ml test volume in 20 ml glass scintillation vials, placed on a microplate shaker or on an ordinary shaking table, but smaller containers and test volumes (down to 1 ml) could also be used. Tissue culture treated polystyrene microplates were found toxic to algae and thus unusable. pH control is achieved more easily in the minitest than in larger size shake flasks due to greater turbulence and a larger surface/volume ratio which both facilitates CO2 mass transfer. Uniform illumination of the individual units of a minitest setup is obtained readily due to the small area that has to be illuminated. Using the rapidly growing green alga S. capricornutum as test organism, it is proposed generally to reduce the standard test duration from 3 days to 2 days (minitest as well as larger volume tests) in order to avoid excessive biomass growth. Shortening tests to 2 days appears necessary if light intensity and temperature are near the upper limits of the intervals stated in the ISO standard.

Removal of organic micropollutants at PPB levels in laboratory activated sludge reactors under various operating conditions : biodegradation

Abstract The biodegradation behaviour of 5 model chemical contaminants, 2,4-dichlorophenoxy acetic acid (2,4-D); 2,4,6-trichlorophenol (TCP); pentachlorophenol (PCP); 4-nitrophenol (4-NP) and lindane, spiked into synthetic peptone sewage to μg/l levels (5–1000 μg/l) have been studied in laboratory activated sludge reactors under a range of operating conditions (sludge ages of 1–32 days and sludge loadings of 0.09–0.9 mg BOD5/mg MLSS/d). The reactors were continuously stirred tanks operated under a computer controlled fill and draw cycle of 4 h. Experiments were started with sludge collected from a municipal treatment plant. In most experiments, a gradual adaptation took place resulting in increases in biodegradation rates by an order of magnitude or more compared to initial biodegradation rates and rates obtained in comparative batch experiments, respectively. Times required for adaptation varied among chemicals and experiments and ranged from 2–5 days for 4-NP to 1–2 months for 2,4-D and lindane. No concentration thresholds for adaptation were observed, and the biodegradation generally followed first-order kinetics. Removals by biodegradation in successfully adapted systems were generally within a range of about 40 to about 95% except for 4-NP, which was degraded to concentration levels below the analytical detection limit. For estimation of the part of the total removal which was due to biodegradation the amounts of PCP, TCP and lindane wasted with the sludge were calculated from sorption isotherms. Volatilization was negligible. The operational parameters influenced the biodegradation differently for the different chemicals. PCP was degraded best at high sludge ages and was not degraded significantly at sludge ages below approx. 8 days. This suggests catabolic degradation by slow growing specific degraders. TCP was consistently degraded at high sludge ages (low sludge loadings) and at random at lower sludge ages, but rate constants were highest at intermediate sludge loadings. A similar pattern was seen for 2,4-D. Lindane was degraded poorly at high sludge ages and to a much greater extent at intermediate and at high sludge loadings. These findings suggest co-oxidation as the dominant degradation mechanism for lindane and as a significant mechanism for TCP and 2,4-D.

Algal tests with soil suspensions and elutriates: A comparative evaluation for PAH contaminated soils

An algal growth inhibition test procedure with soil suspensions is proposed and evaluated for PAH-contaminated soil. The growth rate reduction of the standard freshwater green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum) was used as the toxicity endpoint, and was quantified by measuring the fluorescence of solvent-extracted algal pigments. No growth rate reduction was detected for soil contents up to 20 g/l testing five non-contaminated Danish soils. Comparative testing with PAH-contaminated soil elutriates and soil suspensions showed that the suspensions had toxicity endpoints 2.5-3000 times lower than tests with the corresponding elutriates. Algal growth inhibition tests with soil suspensions are recommended for screening purposes as a supplement to elutriate testing. Experiments with a phenanthrene-spiked soil, showed that the sorbed compound did not contribute to the toxicity. However, the soil did act as a reservoir for phenanthrene, allowing desorption to occur continuously during the algal test which maintained higher concentrations of phenanthrene in the dissolved phase. Phenanthrene-spiked soil incubated for 90 days before algal testing, resulted in a reduction of the toxicity to P. subcapitata by a factor of 76 (from EC10 = 0.3 to 23.6 g soil/l). However, during this 90-day period the total concentration of phenanthrene in the soil decreased by 38% (from 322 to 199 mg/kg) indicating that phenanthrene in the aged soil had become less bioavailable.

Removal of organic micropollutants in laboratory activated sludge reactors under various operating conditions: sorption

Abstract The sorption to activated sludge biomass of four model chemical pollutants, 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), pentachlorophenol (PCP) and lindane, was studied in laboratory experiments. Sorption isotherms were determined for three biomass samples: a sludge sample from a municipal waste/water treatment plant and two resulting laboratory cultures grown on synthetic peptone sewage at solids retention times (SRT) of 2.7 and > 100 days, respectively. The sorption experiments were performed in test tubes with various concentrations of model compounds added. Based on linear sorption coefficients, the removal by sorption was calculated for activated sludge reactors operated at various conditions. This made it possible to quantify that part of the observed removal of the model compounds, which was due to biodegradation, and that part which was due to sorption. Removal by sorption was only important for PCP and was up to 50% of the total removal at short SRTs ( 14 days). There are two reasons to expect that sorption in bioreactors with low SRTs will play a more important role than for bioreactors with high SRTs: (i) at low SRT the slow growing specific degraders will be washed out from the reactor system. Consequently, biodegradation is stopped and the concentration level in the bioreactor increases, which leads to an increased sorption and (ii) a higher mass-flow of wasted biomass will contribute to an increased removal of pollutants in the sorbed phase. The linear sorption coefficients for 2,4-DCP and 2,4,6-TCP were a little higher than expected from log K ow correlations, for PCP much higher than expected and for the non-polar compound lindane within the expected range. For the chlorinated phenols, this was probably due to some sorption of the ionized phenolate fractions.

Dose-response regressions for algal growth and similar continuous endpoints: calculation of effective concentrations.

We derive equations for the effective concentration giving 10% inhibition (EC10) with 95% confidence limits for probit (log-normal), Weibull, and logistic dose-response models on the basis of experimentally derived median effective concentrations (EC50s) and the curve slope at the central point (50% inhibition). For illustration, data from closed, freshwater algal assays are analyzed using the green alga Pseudokirchneriella subcapitata with growth rate as the response parameter. Dose-response regressions for four test chemicals (tetraethylammonium bromide, musculamine, benzonitrile, and 4-4-(trifluoromethyl)phenoxy-phenol) with ranges of representative slopes at 50% response (0.54-2.62) and EC50s (2.20-357 mg/L) were selected. Reference EC50s and EC10s with 95% confidence limits using probit or Weibull models are calculated by nonlinear regression on the whole dataset using a dose-response regression program with variance weighting and proper inverse estimation. The Weibull model provides the best fit to the data for all four chemicals. Predicted EC10s (95% confidence limits) from our derived equations are quite accurate; for example, with 4-4-(trifluoromethyl)phenoxy-phenol and the probit model, we obtain 1.40 (1.22-1.61) mg/L versus 1.40 (1.20- 1.64) mg/L obtained from the nonlinear regression program. The main advantage of the approach is that EC10 or ECx (where x = 1-99) can be predicted from well-determined responses around EC20 to EC80 without experimental data in the low- or high-response range. Problems with the estimation of confidence interval for EClow,x (concentration predicted to cause x% inhibition) from algal growth inhibition also are addressed. Large confidence intervals may be the result of experimental error and lack of a well-defined reference response value.

No-effect concentrations in algal growth inhibition tests

We propose three simple models for effects of chemical compounds on the growth of batch cultures of algae that allow the estimation of the no-effect concentration. The growth model assumes that the costs for growth is proportional to the concentration that exceeds the no-effect level. The hazard model assumes that the hazard rate is proportional to the concentration that exceeds the no-effect level. The adaptation model is similar to the hazard model, but the effects only occur at the start. The no-effect concentrations of the three models turn out to be very similar.

Evaluation of a phytoplankton toxicity test for water pollution assessment and control

A suggested standard test protocol for the short term14C assimilation algal toxicity test method (photosynthesis inhibition test) has been evaluated with natural phytoplankton and cultures of the marine diatomSkeletonema costatum. A number of test technical factors as well as the variability in the sensitivity of natural phytoplankton have been investigated, using potassium dichromate as a reference toxicant in all tests. Some supplementary experiments were carried out with pentachlorophenol and with an industrial effluent. The sensitivity of the test increased with increasing incubation time, but for practical reasons 6 hours of incubation time is recommended.The method showed good reproducibility. The sensitivity of natural phytoplankton, however, varied considerably, both between sampling locations and with time. For testing of effluents it was found necessary to measure the CO2 alkalinity and correct for changes in the specific14C activity caused by the deviating alkalinity of the effluent.Advantages and disadvantages of using natural phytoplankton or cultured algal species in toxicity tests for water pollution assessment and control are discussed. Tests with natural phytoplankton are not suitable for use in effluent control schemes or for similar regulatory purposes because their sensitivity is too variable. However, such tests are considered realistic indicators of the actual acute toxic effects on the phytoplanktonic community of a receiving water body. Laboratory tests with cultures ofSkeietonema costatum are reproducible and can be performed all year round and are therefore well suited for regulatory uses.